Lipid-like nanocomplexes and uses thereof

ABSTRACT

Disclosed are compounds of formula (I) below: 
     
       
         
         
             
             
         
       
         
         
           
             wherein each of the variables A, B, X, W, V, R 1 -R 5 , and m is defined herein. Also disclosed are pharmaceutical compositions containing a nanocomplex, wherein the nanocomplex is formed of one of the compounds, and a protein, a nucleic acid, or a small molecule; and methods of treating a medical condition with one of the pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser.16/966,368, filed Jul. 30, 2020; which is a U.S. National Stage ofInternational Application No. PCT/US2019/016362, filed Feb. 1, 2019;which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 62/625,153, filed Feb. 1, 2018.

GOVERNMENT SUPPORT

This invention was made with government support under grant 1452122awarded by the National Science Foundation, grants EB027170 and TR002636awarded by the National Institutes of Health, and grant N00014-16-1-2550awarded by the United States Navy. The government has certain rights inthe invention.

BACKGROUND

Protein-based therapeutics are used for transient and accuratemanipulation of cell functions because of their high specificities andlow off-target effects. For example, clustered regularly interspacedshort palindromic repeat associated protein 9, i.e., CRISPR/Cas9,demonstrates high flexibility and specificity for genome editing eithervia gene deletion, insertion, activation, and repression or viaepigenetic modification. CRISPR/Cas9 facilitates disease modeling andidentification of new treatments for various genetic disorders andinfectious diseases.

A protein such as CRISPR/Cas9 must be delivered to its target site,i.e., an intracellular target, to achieve therapeutic effects. Yet, ithas been a long-standing challenge to develop safe and efficientcarriers for intracellular delivery of therapeutic proteins.

Conventional methods for delivering proteins include mechanical/physicaltechniques (e.g., microinjection, electroporation, and hydrodynamicinjection) and carrier-based biochemical modifications (e.g., nuclearlocalization signal peptides, lipid or lipid-like nanocomplexes, andpolymeric assemblies). The mechanical/physical techniques, although notrequiring carriers, turn out to be invasive, raising practical issuesfor in vivo application. On the other hand, carriers used in biochemicalmodifications, while capable of delivering proteins intracellularly,exhibit significant limitations, e.g., low transfection efficiency andhigh cytotoxicity.

There is a need to develop a new carrier without the above-mentionedlimitations for delivering a protein to its target site.

SUMMARY

The present invention relates to certain lipophilic compounds forforming lipid-like nanocomplexes that can be used for delivering aprotein, e.g., CRISPR/Cas9, to its target site.

Unexpectedly, these lipid-like nanocomplexes demonstrate highertransfection efficiency and lower cytotoxicity than Lipofectamine 2000(Lpf2k), a commonly used commercial agent for delivering proteins.

In one aspect of this invention, it covers two sets of lipid-likecompounds of formula (I) below:

In one set, referring to formula (I), A is a hydrophilic head selectedfrom

in which each of R_(a), R_(a)′, R_(a)″, and R_(a)′″, independently, isH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₂₀ cycloalkyl,C₁-C₂₀ heteroalkyl, C₁-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and Zis a C₁-C₂₀ bivalent aliphatic radical, a C₁-C₂₀ bivalentheteroaliphatic radical, a bivalent aryl radical, or a bivalentheteroaryl radical; B is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₄ cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl, or

each of R₁ and R₂, independently, is a C₁-C₂₀ bivalent aliphaticradical; each of R₃ and R₄, independently, is H or C₁-C₁₀ alkyl, or R₃and R₄, together with the atom to which they are attached, form C₃-Clocycloalkyl; R₅ is C₁ - C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₄cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl; W is O, S, or Se; V is a bond, O, S, or Se; X, a linker, is

in which each of L₁, L₂, L₃, and L₄, independently, is a bond, O, S, orNR_(c); G is O, S, or NR_(d); Q is OR_(f), SR_(g), or NR_(h)R_(i); andeach of r and t, independently, is 1-6, each of R_(c), R_(d), R_(f),R_(g), R_(h), and R_(i), independently, being H, C₁-C₁₀ alkyl, C₁-C₁₀heteroalkyl, aryl, or heteroaryl; and m is 0 or 1, provided that m is 1when V is S.

In the other set, referring to formula (I) again, A is a hydrophilichead selected from

in which each of R_(a), R_(a)′, R_(a)′″, and R^(a)′″, independently, isH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₂₀ cycloalkyl,C₁-C₂₀ heteroalkyl, C₁-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and Zis a C₁-C₂₀ bivalent aliphatic radical, a C₁-C₂₀ bivalentheteroaliphatic radical, a bivalent aryl radical, or a bivalent ioheteroaryl radical; B is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₄ cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl, or

R₁ is a C₁-C₂₀ bivalent aliphatic radical; R₂ is a bond or C₁-C₂₀bivalent aliphatic radical; each of R₃ and R₄, independently, is H orC₁-C₁₀ alkyl, or R₃ and R₄, together with the atom to which they areattached, form C₃-C₁₀ cycloalkyl; R₅ is

is in which R₆ is a bond or C₁-C₂₀ bivalent aliphatic radical; each ofR_(b) and R_(b)′is F or, R_(b) and R_(b)′, together with the atom towhich they are attached, form C═O; R₇ is F or an aliphatic lipid moiety;each of L₁ and L₂, independently, is a bond, O, S, or NR_(c), R_(c)being H, C₁-C₁₀ alkyl, C₁-C₁₀ heteroalkyl, aryl, or heteroaryl; and n is1 to 20;

each of W and V, independently, is a bond, O, S, or Se; X, a linker, isin which each of L₃, L₄, Ls, and L₆, independently, is a bond, O, S, orNRC; G is O, S, or NR_(d); Q is OR_(f), SR_(g), or NR_(h)R_(i); and eachof r and t, independently, is 1-6, each of R_(c), R_(d), R_(f), R_(g),R_(h), and R_(i), independently, being H, C₁-C₁₀ alkyl, C₁-C₁₀heteroalkyl, aryl, or heteroaryl; and m is 0 or 1.

Typically, the above-described lipid-like compounds have variable A aseither

each of R_(a) and R_(a)′, independently, being a C₁-C₁₀ monovalentaliphatic radical, a C₁-C₁₀ monovalent heteroaliphatic radical, amonovalent aryl radical, or a monovalent heteroaryl radical; and Z beinga C₁-C₁₀ bivalent aliphatic radical, a C₁-C₁₀ bivalent heteroaliphaticradical, a bivalent aryl radical, or a bivalent heteroaryl radical.These compounds preferably have variable B as

The term “lipid-like compounds” herein refers to compounds that containone or more hydrophilic (or polar) amine-containing head groups and oneor more hydrophobic (or nonpolar) hydrocarbon-containing tails. See,e.g., Love et al., PNAS, 2010, 107(5), 1864-1869. The term “lipid-likenanocomplexes” refers to nanocomplexes that contain one of lipid-likecompounds. See, e.g., Wang et al., Angew. Chem. Int. Ed., 2014, 53(11),2893-2898.

The term “aliphatic” herein refers to a saturated or unsaturated, linearor branched, acyclic, cyclic, or polycyclic hydrocarbon moiety. Examplesinclude, but are not limited to, alkyl, alkylene, alkenyl, alkenylene,alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl,cycloalkenylene, cycloalkynyl, and cycloalkynylene moieties.

The term “aliphatic lipid moiety” herein refers to a hydrophobic moietythat contains long-chain, saturated or unsaturated, linear or branched,acyclic, cyclic, or polycyclic hydrocarbons, alcohols, aldehydes, orcarboxylic acids. Examples include, but are not limited to, cholesterol,desmosterol, and lanosterol.

The term “alkyl” or “alkylene” refers to a saturated, linear or branchedhydrocarbon moiety, such as methyl, methylene, ethyl, ethylene, propyl,propylene, butyl, butylenes, pentyl, pentylene, hexyl, hexylene, heptyl,heptylene, octyl, octylene, nonyl, nonylene, decyl, decylene, undecyl,undecylene, dodecyl, dodecylene, tridecyl, tridecylene, tetradecyl,tetradecylene, pentadecyl, pentadecylene, hexadecyl, hexadecylene,heptadecyl, heptadecylene, octadecyl, octadecylene, nonadecyl,nonadecylene, icosyl, icosylene, triacontyl, and triacotylene. The term“alkenyl” or “alkenylene” refers to a linear or branched hydrocarbonmoiety that contains at least one double bond, such as —CH═CH—CH₃ and—CH═CH—CH₂—. The term “alkynyl” or “alkynylene” refers to a linear orbranched hydrocarbon moiety that contains at least one triple bond, suchas —C≡C—CH₃ and —C≡C—CH₂—. The term “cycloalkyl” or “cycloalkylene”refers to a saturated, cyclic hydrocarbon moiety, such as cyclohexyl andcyclohexylene. The term “cycloalkenyl” or “cycloalkenylene” refers to anon-aromatic, cyclic hydrocarbon moiety that contains at least onedouble bond, such as cyclohexenyl cyclohexenylene. The term“cycloalkynyl” or “cycloalkynylene” refers to a non-aromatic, cyclichydrocarbon moiety that contains at least one triple bond, cyclooctynyland cyclooctynylene.

The term “heteroaliphatic” herein refers to an aliphatic moietycontaining at least one heteroatom selected from N, O, P, B, S, Si, Sb,Al, Sn, As, Se, and Ge.

The term “alkoxy” herein refers to an —O-alkyl. Examples of alkoxyinclude methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,sec-butoxy, and tert-butoxy.

The term “aryl” herein refers to a C₆ monocyclic, C₁₀ bicyclic, C₁₄tricyclic, C₂₀ tetracyclic, or C₂₄ pentacyclic aromatic ring system.Examples of aryl groups include phenyl, phenylene, naphthyl,naphthylene, anthracenyl, anthrcenylene, pyrenyl, and pyrenylene. Theterm “heteroaryl” herein refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, 11-14 membered tricyclic, and 15-20 memberedtetracyclic ring system having one or more heteroatoms (such as O, N, S,or Se). Examples of heteroaryl groups include furyl, furylene,fluorenyl, fluorenylene, pyrrolyl, pyrrolylene, thienyl, thienylene,oxazolyl, oxazolylene, imidazolyl, imidazolylene, benzimidazolyl,benzimidazolylene, thiazolyl, thiazolylene, pyridyl, pyridylene,pyrimidinyl, pyrimidinylene, quinazolinyl, quinazolinylene, quinolinyl,quinolinylene, isoquinolyl, isoquinolylene, indolyl, and indolylene.

Unless specified otherwise, aliphatic, heteroaliphatic, alkoxy, alkyl,alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl,cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl,cycloalkynylene, heterocycloalkyl, heterocycloalkylene,heterocycloalkenyl, heterocycloalkenylene, aryl, and heteroarylmentioned herein include both substituted and unsubstituted moieties.Possible substituents on cycloalkyl, cycloalkylene, cycloalkenyl,cycloalkenylene, cycloalkynyl, cycloalkynylene, heterocycloalkyl,heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl,and heteroaryl include, but are not limited to, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₂₀ alkoxy, C₃-C₂₀ cycloalkyl, C₃-C₂₀cycloalkenyl, C₃-C₂₀ heterocycloalkyl, C₃-C₂₀ heterocycloalkenyl, C₁-C₁₀alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C₁-C₁₀alkylamino, C₂-C₂₀ dialkylamino, arylamino, diarylamino, C₁-C₁₀alkylsulfonamino, arylsulfonamino, C₁-C₁₀ alkylimino, arylimino, C₁-C₁₀alkylsulfonimino, arylsulfonimino, hydroxyl, halo, thio, C₁-C₁₀alkylthio, arylthio, C₁-C₁₀ alkylsulfonyl, arylsulfonyl, acylamino,aminoacyl, aminothioacyl, amido, amidino, guanidine, ureido, thioureido,cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxyl, andcarboxylic ester. On the other hand, possible substituents on aliphatic,heteroaliphatic, alkyl, alkylene, alkenyl, alkenylene, alkynyl, andalkynylene include all of the above-recited substituents except C₁-C₁₀alkyl. Cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene,heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl,heterocycloalkenylene, aryl, and heteroaryl can also be fused with eachother.

The lipid-like compounds described above include the compoundsthemselves, as well as their salts and solvates, if applicable. A salt,for example, can be formed between an anion and a positively chargedgroup (e.g., amino) on a lipid-like compound. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate,fumurate, glutamate, glucuronate, lactate, glutarate, and maleate.Likewise, a salt can also be formed between a cation and a negativelycharged group (e.g., carboxylate) on a lipid-like compound. Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion. The lipid-likecompounds also include those salts containing quaternary nitrogen atoms.A solvate refers to a complex formed between a lipid-like compound and apharmaceutically acceptable solvent. Examples of pharmaceuticallyacceptable solvents include water, ethanol, isopropanol, ethyl acetate,acetic acid, and ethanolamine

Another aspect of this invention relates to a pharmaceutical compositioncontaining a nanocomplex formed of a lipid-like compound described aboveand and a protein or a nucleic acid. In this composition, thenanocomplex has a particle size of 50 to 1000 nm (e.g., 50 to 500 nm, 50to 300 nm, and 50 to 180 nm). The lipid-like compound binds to theprotein or nucleic acid via a non-covalent interaction, a covalent bond,or both.

The term “protein” refers to a polymer of natural or non-natural aminoacids linked together by amide bonds and having a molecular weight of800 Dalton or higher. The term “nucleic acid” refers to a polymer ofnucleotides linked together by phosphodiester bonds, having a molecularweight of 800 Dalton or higher. Both of these polymers can be chemicallymodified. Examples of protein modification include PEGylation andcarboxylation of amine groups in lysine residues contained therein. Morespecifically, carboxylation of proteins or peptides can be achieved byusing cis-aconitic anhydride. See Lee et al., Angew. Chem. Int. Ed.,2009, 48, 5309-5312; Lee et al., Angew. Chem. Int. Ed., 2010, 49,2552-2555; and Maier et al., Journal of the American Chemical Society,2012, 134, 10169-10173.

The term “non-covalent interaction” refers to any non-covalent binding,which includes ionic interaction, hydrogen bonding, van der Waalsinteraction, and hydrophobic interaction.

The pharmaceutical composition typically contains a pharmaceuticallyacceptable carrier. The carrier in the pharmaceutical composition mustbe “acceptable” in the sense that it is compatible with the activeingredient of the composition (and preferably, capable of stabilizingthe active ingredient) and not deleterious to the subject to be treated.One or more solubilizing agents can be utilized as pharmaceuticalexcipients for delivery of an active glycoside compound. Examples ofother carriers include colloidal silicon oxide, magnesium stearate,cellulose, sodium lauryl sulfate, and D&C Yellow #10.

Further covered by this invention is a method of treating a medicalcondition, e.g., a lung disease. The method includes a step ofadministering to a subject in need thereof an is effective amount of anabove-described pharmaceutical composition.

The details of the invention are set forth in the description below.Other features, objects, and advantages of the invention will beapparent from the following drawings and detailed description of severalembodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of synthesis of lipid-like compounds(lipidoids) and encapsulation of proteins into lipidoid nanoparticles.(a) Encapsulation of negatively charged GFP-Cre and Cas9:sgRNA intosynthetic cationic lipidoid nanoparticles (LNPs) for intracellularprotein delivery and genome editing. (b) Synthetic route and lipidoidsnomenclature. (c) Chemical structures of amine heads for lipidoidssynthesis.

FIG. 2 is a schematic depiction of characterization of lipidoids andLNPs. (a) and (b) ¹H NMR and ESI-MS spectra of 76-O17O, 76-O17S, and76-O17Se (see exemplary lipid-like compounds below). (c) Statisticalanalysis of averaged hydrodynamic diameter (<D_(h)>) distribution ofLNPs. (d) Typical hydrodynamic diameter distributions of 76-O17O,76-O17S, and 76-O17Se LNPs.

FIG. 3 is a schematic depiction of another characterization of lipidoidsand LNPs. (a) and (b) Typical transmission electron microscopy (TEM)images and relative size variations of 76-O17O, 76-O17S, and 76-O17SeLNPs (scale bar being 100 nm). (c) Fluorescent emission intensities andFRET ratios of DiO/DiI loaded 76-O17Se LNPs during storage.

FIG. 4 is a schematic depiction of in vitro screening of LNPs forprotein delivery. (a) Typical images of (−30)GFP-Cre protein and(−30)GFP-Cre loaded 76-O17O, 76-O17S, and 76-O17Se LNPs treatedHeLa-DsRed cells. Scale bar=200 μm. (b) Percentage of GFP-positive cellsshown for 51 LNPs tested. Data points marked in red for LNPs inducedhigh level of transfection. (c) The tails O17O, O17S, and O17Se)influenced (−30)GFP-Cre protein transfection activity.

FIG. 5 is a schematic depiction of structure-activity relationship forLNPs. (a) and (b) Apparent pKa values and phospholipid bilayer membranedisruption ability influenced (-protein delivery efficiency. (c)Relative hit rates of efficacious LNPs having none, one, or twoproperties. (d) Relative hit rates of efficacious LNPs having O17O,O17S, or O17Se tails.

FIG. 6 shows the efficiency of (−30)GFP-Cre delivery with LNPs. (a)DsRed is expression of HeLa-DsRed cells treated with (−30)GFP-Cre and(−30)GFP-Cre loaded LNPs. (b) Cell viability of HeLa-DsRed cells treatedwith (−30)GFP-Cre and (−30)GFP-Cre loaded LNPs

FIG. 7 is a schematic depiction of typical fluorescence images ofsections of lungs obtained from Ai14 mice treated with PBS andGFP-Cre/LNPs (the first column being 4′,6-diamidino-2-phenylindole orDAPI; the second column being tdTomato; the third column shwoing themerging of the first and second columns; and scale bar being 100 μm).

FIG. 8 shows the efficiency of Cas9:sgRNA delivery with LNPs. (a) GFPknockout of GFP-HEK cells treated with Cas9:sgRNA and Cas9:sgRNA/LNPs.(b) Cell viability of GFP-HEK cells treated with Cas9:sgRNA andCas9:sgRNA/LNPs.

FIG. 9 is a schematic presentation of cholesterol-based andreduction-responsive combinatorial lipidoids for intracellular delivery.(A) Chemical structures of cationic lipidoids and amine head groups. (B)Lipidoids nanoparticles as a versatile platform for anticancer drugs,mRNA and protein delivery.

FIG. 10 shows the characterization of lipioids and nanoparticles. (A)MALDI-TOF spectra of lipidoids. (B) Hydrodynamic diameter andpolydispercity of lipidoid nanoparticles measured by DLS. (C) TEM imagesof lipidoid nanoparticles. Scal bar=200 nm. (D) Relative size change ofblank nanoparticles under storage. (E) Cytotoxicity tests of OcholB,O16B and Lpf2k nanoparticles. P<0.05, student's t-test.

FIG. 11 shows the thiol-triggered morphological variation and cargorelease. (A) Time-dependent relative size variation of the lipidoidnanoparticles with DTT and Cysteine treatment. (B) TEM images oflipidoid nanoparticles treated with DTT. Scale bar=600 nm. (C) Relativesize change of lipidoid nanoparticle after 24 h of DTT treatment. (D)Fluorescent emission spectra of cargoes loaded nanoparticles. (E)Time-dependent NR release profile. (F) Fluorescent intensity of calceinencapsulated lipidoid nanoparticles treated with DTT or Cysteine. (G)RNA binding test of lipidoid nanoparticles with and without DTTtreatment.

FIG. 12 shows the internalization study of cargo-loaded lipidoidnanoparticles. (A) Time-dependent FRET ratio variation of DiO-DiI loadednanoparticles. (B) Time-dependent NR⁺ cells portions of HeLa cellstreated with NR loaded nanoparticles. (C) NR⁺ cells portions of lipidoidnanoparticles after 8 h of exposure. (D) Fluorescent images of HeLacells treated by NR loaded nanoparticles. Scale bar=100 μm. (E) Meanfluorescent intensity of HeLa cells treated with free or nanoparticlesencapsulated calcein. (F) Transfection efficiencies of (−30)GFP-Creprotein by lipidoid nanoparticles against HeLa-DsRed cells. (G) Meanfluorescent intensity, (H) flow cytometry histogram, (I) fluorescentimages and (J) bright field images of (−30)GFP-Cre/LNPs treatedHeLa-DsRed cells. Scale bar=110 μm.

FIG. 13 shows the intracellular delivery of anticancer drugs. (A)Absorption and fluorescent emission spectra of CPT and Dox loadednanoparticles. (B) Mean fluorescent intensity of free and nanoparticleencapsulated Dox treated HeLa cells. (C) Dose-dependent cytotoxicity offree Dox, and blank and Dox loaded nanoparticles. (D) Cytotoxicity offree and nanoparticle encapsulated CPT and Oxa.

FIG. 14 shows the intracellular delivery of mRNA. (A) LNP/mRNA weightratio and (B) mRNA dose-dependent transfection efficacy. (C) Fluorescentimages of mRNA/LNPs treated HeLa cells. Scale bar=100 μm. (D)Transfection efficiency and (E) cytotoxicity of mRNA/LNPs. (F) Brightfield images of mRNA/LNPs treated HeLa cells. Scale bar=110 μm. (G) CremRNA and (H) Cas9 mRNA and sgRNA delivery by OCholB LNPs.

FIG. 15 shows the intracellular delivery of genome editing protein. (A)Internalization mechanism study. (B) Genome editing efficiency, (C) flowcytometry histogram, (D) cytotoxicity and (E) bright field images of(−30)GFP-Cre/LNPs treated HeLa-DsRed cells. Scale bar=200 μm. (F) Genomeediting efficacy was plotted against cell viability for each testedconditions.

FIG. 16 shows the In vivo toxicity tests. (A) Time-dependent body weightand (B) biochemical blood analysis of blank LNPs injected Balb/c mice.

FIG. 17 shows the mRNA and protein delivery for in vivo genome editingusing adult Ai14 mice. (A) Cre-mediated gene recombination. Protocolsused for (B) intramuscular and (C) intravenous injections. Fluorescentimages of (D) intramuscular protein/LNPs (scale bar=270 μm) and (E)intramuscular mRNA/LNPs (scale bar=270 μm) injected skeletal muscles.Fluorescent images of (F) lungs from control and intravenousprotein/LNPs injected mice (scale bar=135 μm) and (G) spleens fromintravenous mRNA/LNPs injected mice (scale bar=190 μm). Red channel inthe original image, tdTomato; Blue channel in the original image, DAPI.Images in up panels are from nanoparticles injected mice and images inlow panels are from untreated control mice.

FIG. 18 is a schematic presentation showing (a) Encapsulation of AmBinto synthetic cationic lipidoids nanoparticles and effect on funguscells. (d) The quaternized lipidoids were combinatorial synthesized ofthe amine and alkyl-eposxide molecules, lipidoids are named asfollows(Carbon numbers of tail)-(Amine number)

FIG. 19 shows the visual stability states after preparation:AmB/(75-O4B, 78-O14B, 87-O14B) encapsulates demonstrated opaquesuspention and all precipitated within 1 week, AmB/(75-O14B, 78-O14B,87-O14B)-F encapsulates demonstrated translucent solutions afterpreparation and not homogenize at the end of 2 week, AmB/(Q75-O14B,Q78-O14B, Q87-O14B) and AmB/(Q75-O14B, Q78-O14B, Q87-O14B) -Fencapsulates exhibit homogenous transparent yellow solutions and stablein the following 2 weeks.

FIG. 20 shows (a) Hydrodynamic sizes of AmB encapsulates and Fungizone®after preparation and in following 2 weeks(n=9), (b) Polydispersityindexes of AmB encapsulates after preparation and in following 2 weeksdetermined by DLS(n=9). *p<0.05 and ** p<0.001vs the particle size andPDI after preparation.

FIG. 21 shows (a) The DLC of AmB encapsulates and Fungizone® (n=3). (b)The MIC of different AmB encapsulates, free AmB and Fungizone® againstCandida. albicans(SC₅₃₁₄) after 24 h and 48 h incubation from range of14.0 to 0.109375 μg/mL (n=9), *P<0.05 vs Fungizone®.

FIG. 22 shows (a) Hemolysis of human RBCs by AmB encapsulates, free AmBand Fungizone® at equivalent of AmB concentrations (25, 50, 100, 200μg/mL) after 1 h incubation at 37° C(n=9). (b) In vitro MTT test ofdifferent AmB encapsulates, free AmB and Fungizone® towards HEK293 cellline at equivalent of AmB concentrations (25, 50, 100, 200 μg/mL) after24 h incubation. All data present as mean±SD (n=9), *p<0.05 and**p<0.001 vs Fungizone®

FIG. 23 shows (a) Plasma concentrations of AmB after intravenousinjection of AmB/Q78-O14B-F and Fungizone® at a dose of 2 mg AmB/kg toSD rats; (b-e) The AmB concentrations in mice tissues after 48 h and 72h intravenous treatment of AmB/Q78-O14B-F (10 mg, 5 mg, 2 mg AmB/kg,respectively) and Fungizone® (2 mg AmB/kg) by HPLC, (b) Liver,(c)Spleen, (d) Lung, (e) Kidney, *p<0.05 and **p<0.001 vs Fungizone®.

FIG. 24 shows the In vivo toxicity of (a) Creatinine, (b) BUN, (c) ALT,(d) AST level in healthy BALB/c mice 48 h and 72 h after intravenousadministrated with AmB/Q78-O14B-F at dose of 10 mg, 5 mg and 2 mg AmB/kgand Fungizone® at dose of 2 mg AmB/kg (n=3), *p<0.05 and **p<0.001 vsFungizone®.

FIG. 25 is a bar graph showing the percentage of GFP positive cells(GFP+) as a function of the fluorine-containing lipidoid used forprotein delivery.

FIG. 26 is a bar graph showing the percentage of DsRed positive cells asa function of the fluorine-containing lipidoid used for proteindelivery.

FIG. 27 is a bar graph showing the percentage of GFP+ cells as afunction of the lipidoid (derived from lipids with different hydrophobictails and synthesized from amine 200) used for protein delivery.

FIG. 28 is a bar graph showing the percentage of GFP+ cells as afunction of the lipidoid (derived from lipids synthesized from differentcyclic amine analogues) used for protein delivery for.

FIG. 29 is a bar graph showing the observed luminescence stemming frommRNA delivery to CD8+ T cells as a function of the lipidoid (derivedfrom lipids synthesized from different imidazole-containing amineanalogues).

DETAILED DESCRIPTION

Disclosed in detail herein are lipid-like compounds of the presentinvention. More specifically, two embodiments are described in orderbelow.

In the first embodiment, referring to formula (I) shown above, A is ahydrophilic head selected from

in which each of R_(a), R_(a)′, R_(a)′″, and R_(a)′″, independently, isH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₂₀ cycloalkyl,C₁-C₂₀ heteroalkyl, C₁-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and Zis a C₁-C₂₀ bivalent aliphatic radical, a C₁-C₂₀ bivalentheteroaliphatic radical, a bivalent aryl radical, or a bivalentheteroaryl radical; B is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₄ cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl, or

each of R₁ and R 2 , independently, is a C₁-C₂₀ bivalent aliphaticradical; each of R₃ and R₄, independently, is H or C₁-C₁₀ alkyl, or R₃and R₄, together with the atom to which they are attached, form C₃-C₁₀cycloalkyl; R₅ is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₄cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl; W is O, S, or Se; V is a bond, O, S, or Se; X, a linker, is

in which each of L₁, L2, L₃, and L₄, independently, is a bond, O, S, orNR C ; G is O, S, or NR_(d); Q is OR_(f), SR_(g), or NR_(h)R_(i); andeach of r and t, independently, is 1-6, each of R_(c), R_(d), R_(f),R_(g), R_(h), and R, independently, being H, C₁-C₁₀ alkyl, C₁-C₁₀heteroalkyl, aryl, or heteroaryl; and m is 0 or 1, provided that m is 1when V is S. is This embodiment preferably includes compounds thattypically have variable A as

and variable B as

Exemplary compounds have variables A, B, and R₁-R₅ as follows: A is

B is

each of R₁ and R 2 , independently, is a C₁-C₄ bivalent aliphaticradical; each of R₃ and R₄, independently, is H or C₁-C₄ alkyl; and R₅is Cl-C₂₀ alkyl.

Preferably, A is an amino moiety formed from one of the following amines

As described above, X is a linker. Examples of X include, but are notlimited to,

each of R_(c) and R_(d), independently, being H or C₁-C₁₀ alkyl. Thesecompounds preferably have each of R₁ and R₂, independently, as a C₁-C₄bivalent aliphatic radical; each of R₃ and R₄, independently, as H orC₁-C₄ alkyl; and R₅ as C₁-C₂₀ alkyl.

Turning to variables W, V, and m, this embodiment can include, based onthese three variables, the following three subsets of compounds.

Subset (i) includes the compounds of formula (I), in which each of W andV, independently, is O or Se; and m is 0.

This subset of compounds can have their

moiety formed from one of the following molecules:

in which q is an integer of 8-12.

Subset (ii) includes the compounds of formula (I), in which W is O, S,or Se; V is a bond; and m is 0 or 1.

This subset of compounds can have their

moiety formed from one of the following molecules:

in which q is an integer of 8-12.

Subset (iii) includes the compounds of formula (I), in which each of Wand V is O or to S and m is 1.

This subset of compounds can have their

moiety formed from one of the following molecules:

in which q is an integer of 8-12.

Alternatively, this subset of compounds can have their

moiety formed from one of the following molecules:

in which X is O, S, or NH; R is H or Me; p is an integer of 0-3; q is aninteger of 1-16; and v is an integer of 1-10.

In the second embodiment, referring to the above formula (I) again, A isa hydrophilic head selected from

in which each of R_(a), R_(a)′, R_(a)″, and R_(a)′″, independently, isH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₂₀ cycloalkyl,C₁-C₂₀ heteroalkyl, C₁-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and Zis a C₁-C₂₀ bivalent aliphatic radical, a C₁-C₂₀ bivalentheteroaliphatic radical, a bivalent aryl radical, or a bivalentheteroaryl radical; B is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,C₃-C₂₄ cycloalkyl, C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, orheteroaryl, or

R₁ is a C₁-C₂₀ bivalent aliphatic radical; R₂ is a bond or C₁-C₂₀bivalent aliphatic radical; each of R₃ and R₄, independently, is H orC₁-C₁₀ alkyl, or R₃ and R₄, together with the atom to which they areattached, form C₃-C₁₀ cycloalkyl; R₅ is

in which R₆ is a bond or C₁-C₂₀ bivalent aliphatic radical; each ofR_(b) and R_(b)′ is F or, R_(b) and R_(b)′, together with the atom towhich they are attached, form C═O; R₇ is F or an aliphatic lipid moiety;each of L₁ and L₂, independently, is a bond, O, S, or NR_(c), R_(c)being H, C₁-CE) alkyl, C₁-C₁₀ heteroalkyl, aryl, or heteroaryl; and n is1 to 20; each of W and V, independently, is a bond, O, S, or Se; X, alinker, is

in which each of L₃, L₄, L₅, and L₆, independently, is a bond, O, S, orNR_(c); G is O, S, or NR_(d); Q is OR_(f), SR_(g), or NR_(h)R_(i); andeach of r and t, independently, is 1-6, each of R_(c), R_(d), R_(e),R_(f), R_(g), R_(h), and R_(i), independently, being H, C₁-C₁₀ alkyl,C₁-C₁₀ heteroalkyl, aryl, or heteroaryl; and m is 0 or 1.

Like the first embodiment, the second embodiment can also includecompounds having variable A as

and variable B as

An exemplary compound of this embodiment has variables A, B, and R₁-R₄as

follows: A is

B is

each of R₁ and R₂, independently, is a C₁-C₄ bivalent aliphatic radical;and each of R₃ and R₄, independently, is H or C₁-C₄ alkyl.

Again, A can be an amino moiety formed from one of the following amines

In the second embodiment, examples of X include, but are not limited to,

each of R_(c) and R_(d), independently, being H or C₁-C₁₀ alkyl. Thesecompounds preferably have each of R₁ and R₂ as a C₁-C₄ bivalentaliphatic radical; each of R₃ and R₄, independently, as H or C₁-C₄alkyl; and R₅ as C₁-C₂₀ alkyl.

As to variables W, V, and m, the second embodiment can include compoundshaving each of R₂, W, and V as a bond, and m as 0.

Referring to variable R₅, i.e., compounds in this embodiment can haveeach of L₁ and L₂ as a bond, and each of R_(b), R_(b)′, and R₇ as F.

Exemplary compounds have their one of the following molecules:

moiety formed from

in which j is an integer of 0-10 and k is an integer of 1-20.

Alternatively, this embodiment includes those compounds, in which R₆ isC₁-C₄ io bivalent aliphatic radical; each of L₁ and L₂, independently,is 0 or NR_(c), R_(c) being H or C₁-C₁₀ alkyl; R_(b) and R_(b)′,together with the atom to which they are attached, form C═O; n is 1 or2; and R₇ is an aliphatic lipid moiety. The aliphatic lipid moiety canbe cholesterol.

moiety formed from one of the following molecules:

Exemplary compounds have their

in which X is O or NH and W is O, S, or Se.

The lipid-like compounds of this invention can be prepared by methodswell known in the art. See, e.g., Wang et al., ACS Synthetic Biology,2012, 1, 403-407; Manoharan et al., WO 2008/042973; and Zugates et al.,U.S. Pat. No. 8,071,082.

The synthetic route shown below exemplifies synthesis of certainlipid-like compounds described above:

in which each of variables R_(a), R₂ -R₅ , X, W, V, and m are definedabove.

In this exemplary synthetic route, an amine compound, i.e., compound D,reacts with to a vinyl carbonyl compound E to afford the final product,i.e., compound F Amino compound D can be one of the above-describedCompounds 10, 17, 63, 75-78, 80-82, 87, 90, 93, 103, 304, 306, and 400.

Other lipid-like compounds of this invention can be prepared using othersuitable starting materials through the above-described synthetic routeand others known in the art. The method set forth above can include anadditional step(s) to add or remove suitable protecting groups in orderto ultimately allow synthesis of the lipid-like compounds. In addition,various synthetic steps can be performed in an alternate sequence ororder to give the desired material. Synthetic chemistry transformationsand protecting group methodologies (protection and deprotection) usefulin synthesizing applicable lipid-like compounds are known in the art,including, for example, R. Larock, Comprehensive Organic Transformations(2^(nd) Ed., VCH Publishers 1999); P. G. M. Wuts and T. W. Greene,Greene's Protective Groups in Organic Synthesis (4th Ed., John Wiley andSons 2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis (2^(nd) ed., John Wileyand Sons 2009) and subsequent editions thereof.

Certain lipid-like compounds may contain a non-aromatic double bond andone or more asymmetric centers. Thus, they can occur as racemates andracemic mixtures, single enantiomers, individual diastereomers,diastereomeric mixtures, and cis- or trans- isomeric forms. All suchisomeric forms are contemplated.

As mentioned above, these lipid-like compounds are useful for deliveryof proteins or nucleic acids. They can be preliminarily screened fortheir efficacy in delivering pharmaceutical agents by an in vitro assayand then confirmed by animal experiments and clinic trials. Othermethods will also be apparent to those of ordinary skill in the art.

Not to be bound by any theory, the lipid-like compounds facilitatedelivery of proteins or nucleic acids by forming complexes, e.g.,nanocomplexes and microparticles. The hydrophilic head of such alipid-like compound, positively or negatively charged, binds to a moietyof a protein or nucleic acid that is oppositely charged and itshydrophobic moiety binds to a hydrophobic moiety of the protein ornucleic acid. Either binding can be covalent or non-covalent.

The above described complexes can be prepared using procedures describedin publications such as Wang et al., ACS Synthetic Biology, 2012, 1,403-407. Generally, they are obtained by incubating a lipid-likecompound and a protein or nucleic acid in a buffer such as a sodiumacetate buffer or a phosphate buffered saline (“PBS”).

Further covered by this invention is a pharmaceutical compositioncontaining a nanocomplex formed of a lipid-like compound described aboveand and a protein or a nucleic acid. Again, the lipid-like compoundbinds to the protein or nucleic acid via a non-covalent interaction, acovalent bond, or both.

Examples of the protein or nucleic acid include, but are not limited to,clustered regularly interspaced short palindromic repeat associatedprotein 9 (CRISPR/Cas9), Cre recombinase ((−30)GFP-Cre), andCas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) or Cas9:sgRNARNP.

Still within the scope of this invention is a method of treating amedical condition, e.g., a lung disease, with the above-describedpharmaceutical composition. The method includes administering to asubject (e.g., a patient) in need thereof an effective amount of thepharmaceutical composition.

The term “an effective amount” refers to the amount of complexes that isrequired to confer a therapeutic effect on the treated subject.Effective doses will vary, as recognized by those skilled in the art,depending on the types of diseases treated, route of administration,excipient usage, and the possibility of co-usage with other therapeutictreatment.

To practice the method of the present invention, a composition havingthe above-described complexes can be administered parenterally, orally,nasally, rectally, topically, or buccally. The term “parenteral” as usedherein refers to subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordiglycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their to polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,carboxymethyl cellulose, or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art.

A composition containing the nanocomplexes can also be administered inthe form of suppositories for rectal administration.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific examples are therefore to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

Methods and Materials

General

All chemicals used for lipidoids synthesis were purchased fromSigma-Aldrich without further purification unless otherwise noted.(−30)GFP-Cre recombinase, S. pyogenes Cas9 (spCas9) and sgRNA weregenerated following the protocols reported in Wang at al.,

Proc. Natl. Acad. Sci. USA, 2016, 113, 2868-2873 (“Wang”). HeLa-DsRedand GFP-HEK cells were cultured in Dulbecco's modified eagle's medium(DMEM, Sigma-Aldrich) with 10% fetal bovine serum (FBS, Sigma-Aldrich)and 1% penicillin-streptomycin (Gibco). All ¹H NMR spectra were recordedon a Bruker AVIII 500 MHz NMR spectrometer operated in the Fouriertransform mode. Hydrodynamic size and polydispersity index ofnanoparticles were measured by Zeta-PALS particle size analyzer(Brookhaven Instruments). The apparent pKa values of lipidoids weredetermined using 2-(p-toluidinynaphthalene-6-sulphonic acid) (TNS,Sigma-Aldrich) as fluorescent probe following the protocols reported inHeyes et al., J. Controlled Release, 2005, 107, 276-287. TEMmeasurements were performed on a FEI Technai Transmission ElectronMicroscope. Fluorescence images of tissue slices were obtained usingBZ-X Analyzer fluorescence microscope.

Synthesis of Lipid-Like Compounds (i.e., lipidoids)

Head amines (Sigma-Aldrich) were mixed with acrylates tails (e.g., O17O,O17S, and O17Se) at a molar ratio of 1:2.4 in teflon-lined glassscrew-top vials for 48 hours at 70° C. The crude products were purifiedusing a Teledyne Isco Chromatography system.

One class of lipid-like compounds of formula (I) were synthesized byfollowing the synthetic route shown below:

Head amines R_(a)-NH₂ shown in the above scheme were selected fromCompounds 10, 17, 63, 75-78, 80-82, 87, 90, 93, 103, 304, 306, and 400.

Shown in the table below are the codes, chemical formulas, andanalytical data (ESI-MS) of 51 exemplary lipid-like compounds(“lipidoids”) of formula (I). Note that each lipidoid is coded asX-O17Y, in which X represents the number of an amino compound and Yrepresents O, S, or Se. Code X-O17Y indicates that a lipidoid is formedfrom an amine of Compound X and a lipid molecule of O17Y (Y being O, S,or Se).

For example, lipidoid 10-O17O is formed from amine Compound 10 and lipidmolecule O17O as follows:

Each code in the table below contains O17O, O17S, or O17Se, whichrepresents one of the three molecules:

Lipidoid Code Chemical Formula Cal. [M + H]⁺ Found [M + H]⁺  10-O17OC₄₁H₈₂NO₇ 700.61 700.70  10-O17S C₄₁H₈₂NO_(5S2) 732.56 732.59  10-O17SeC₄₁H₈₂NO₅Se₂ 828.45 828.27  17-O17O C₄₈H₈₈NO₈ 806.65 806.63  17-O17SC₄₈H₈₈NO₆S₂ 838.60 838.49  17-O17Se C₄₈H₈₈NO₆Se₂ 934.49 934.27  63-O17OC₄₃H₈₇N₂O₆ 727.66 727.67  63-O17S C₄₃H₈₇N₂O₄S₂ 759.61 759.62  63-O17SeC₄₃H₈₇N₂O₄Se₂ 855.50 855.35  75-O17O C₄₄H₈₉N₂O₆ 741.67 741.71  75-O17SC₄₄H₈₉N₂O₄S₂ 773.63 773.69  75-O17Se C₄₄H₈₉N₂O₄Se₂ 869.51 869.67 76-O17O C₄₄H₈₇N₂O₆ 739.66 739.74  76-O17S C₄₄H₈₇N₂O₄S₂ 771.61 771.69 76-O17Se C₄₄H₈₇N₂O₄Se₂ 867.50 867.46  77-O17O C₄₅H₈₉N₂O₆ 753.67 753.75 77-O17S C₄₅H₈₉N₂O₄S₂ 785.63 785.64  77-O17Se C₄₅H₈₉N₂O₄Se₂ 881.51881.46  78-O17O C₄₂H₈₅N₂O₆ 713.64 713.79  78-O17S C₄₂H₈₅N₂O₄S₂ 745.59745.57  78-O17Se C₄₂H₈₅N₂O₄Se₂ 841.48 841.43  80-O17O C₄₃H₈₇N₂O₆ 727.66727.68  80-O17S C₄₃H₈₇N₂O₄S₂ 759.61 759.70  80-O17Se C₄₃H₈₇N₂O₄Se₂855.50 855.46  81-O17O C₄₅H₉₁N₂O₆ 755.69 755.71  81-O17S C₄₅H₉₁N₂O₄S₂787.64 787.64  81-O17Se C₄₅H₉₁N₂O₄Se₂ 883.53 883.45  82-O17O C₄₅H₈₉N₂O₆753.67 753.88  82-O17S C₄₅H₈₉N₂O₄S₂ 785.63 785.70  82-O17SeC₄₅H₈₉N₂O₄Se₂ 881.51 881.42  87-O17O C₄₅H₉₁N₂O₈ 787.68 787.71  87-O17SC₄₅H₉₁N₂O₆S₂ 819.63 819.52  87-O17Se C₄₅H₉₁N₂O₆Se₂ 915.52 915.39 90-O17O C₄₄H₈₇N₂O₇ 755.65 755.96  90-O17S C₄₄H₈₇N₂O₅S₂ 787.61 787.59 90-O17Se C₄₄H₈₇N₂O₅Se₂ 883.49 883.38  93-O17O C₄₄H₈₄N₃O₆ 750.64 750.69 93-O17S C₄₄H₈₄N₃O₄S₂ 782.59 782.69  93-O17Se C₄₄H₈₄N₃O₄Se₂ 878.48878.41 103-O17O C₄₄H₈₉N₂O₈ 773.66 773.72 103-O17S C₄₄H₈₉N₂O₆S₂ 805.62805.53 103-O17Se C₄₄H₈₉N₂O₆Se₂ 901.50 901.45 304-O17O C₆₆H₁₃₃N₄O₉1126.01 1125.97 304-O17S C₆₆H₁₃₃N₄O₆S₃ 1173.94 1173.88 304-O17SeC₆₆H₁₃₃N₄O₆Se₃ 1317.77 1317.63 306-O17O C₈₃H₁₆₄N₃O₁₂ 1395.23 1395.24306-O17S C₈₃H₁₆₄N₃O₈S₄ 1459.14 1459.89 306-O17Se C₈₃H₁₆₄N₃O₈Se₄ 1650.921650.77 400-O17O C₆₅H₁₃₀N₃O₉ 1096.98 1096.90 400-O17S C₆₅H₁₃₀N₃O₆S₃1144.91 1144.74 400-O17Se C₆₅H₁₃₀N₃O₆Se₃ 1288.74 1288.60

Another class of lipid-like compounds of formula (I) were synthesized byfollowing the synthetic route shown below:

Again, head amines R_(a)-NH₂ shown in this above scheme were selectedfrom Compounds 10, 17, 63, 75-78, 80-82, 87, 90, 93, 103, 304, 306, and400.

Still another class of lipid-like compounds of formula (I) weresynthesized by following the synthetic route shown below:

Head amines R_(a)-NH₂ shown in the above scheme were selected fromCompounds 10, 17, 63, 75-78, 80-82, 87, 90, 93, 103, 304, 306, and 400.

Fabrication of Nanocomplexes from Lipidoids and Proteins.

Lipidoids were fabricated into nanoparticles for delivery proteins ornucleic acids. Briefly, lipidoids were mix with sodium acetate buffer(25 mM, pH 5.2), sonicated for 30 min in ultrasonic bath and followed byanother 30 mM of vigorous vortex to form lipid-like nanoparticles orLNPs. The LNPs thus obtained were stored at 4° C. For protein/LNPcomplexation, LNPs were mixed with (−30)GFP-Cre or Cas9:sgRNA in PBSbuffer (25 mM, pH 7.4) following the protocols reported in Wang andincubated at room temperature for 30 minutes.

Evaluation of Phospholipid Bilayer Membrane Disruption

Human red blood cells (hRBCs) were washed with PBS buffer three timesand collected after centrifugation at 1000 rpm for 5 minutes. Theresulting stock solution (about 10% v/v hRBCs) was diluted 3 fold in PBSbuffer to give the assay solution. 90 μL of assay solution was mixedwith 10 μL of LNPs solutions (final concentration of lipidoids=3.3 mg/L)and incubated at 37° C. for 60 minutes. Then the samples werecentrifuged again at 1000 rpm for 10 min 10 μL of the supernatant wasfurther diluted into 90 μL of PBS buffer, and the absorbance at 405 nm(0D405) was recorded using a microplate reader. The PBS buffer andTriton X-100 (1% v/v) were used as negative and positive controlsrespectively.

Intracellular Delivery of (−30)GFP-Cre/LNP

For the intracellular uptake study, HeLa-DsRed cells were seeded in48-well plate with a density of 2×10⁴ cell/well. After 24 h ofincubation at 37° C., 5% CO2, (−30)GFP-Cre/LNP nanoparticles were addedto the cells and incubated for 6 h before fluorescence microscopy andflow cytometry (BD FACS Calibur, BD Science, CA) analysis (greenemission from GFP). The final (−30)GFP-Cre protein concentration is 25nM, and lipidoid concentration is 3.3 mg/L. For the gene recombinationfunctional study, HeLa-DsRed cells were treated with same conditions andthe red fluorescence emission from DsRed was analyzed by flow cytometry24 h after delivery.

Intracellular delivery of Cas9:sgRNA/LNP

For CRISPR/Cas9 gene knockout study, GFP-HEK cells were seeded in48-well plate with a density of 2×10⁴ cell/well. After 24 h ofincubation, Cas9:sgRNA/LNP nanoparticles were added to the cells andincubated for 4 h, followed by media changed. After 48 h of incubation,the green emission from GFP was analyzed by flow cytometry. The finalCas9:sgRNA RNP concentration was 25 nM, and lipidoid concentration was3.3 mg/L.

In Vitro Cytotoxicity Assay.

Cell viability was measured by the standard MTT assay. HeLa-DsRed orGFP-HEK cells were seeded into 96-well plate with a density of 5×10³cell/well. (−30)GFP-Cre/LNP or Cas9:sgRNA/LNP nanoparticles were addedafter 24 h of incubation. The final concentration of protein is 25 nMand LNP is 3.3 mg/L. After incubating for 24 h or 48 h, the MTT reagent(5 mg/mL, in 30 μL PBS buffer) was added and the cells were incubatedfor another 4 h at 37° C. The cell culture media were then carefullyremoved and 200μL of DMSO were added. The DMSO solution was transferredinto another 96-well plate and the absorbance at 570 nm was recorded bymicroplate reader. All experiments were performed in quadruplicate.

In Vivo Protein Delivery to Ai14 Mouse

Formulated LNPs (lipidoid/Cholesterol/DOPE/DSPE-PEG2k=16/4/1/4, weightratio) were prepared for protein loading and mice injection. Ai14 micewere housed in a temperature and humidity controlled facility with a 12h light/dark cycle. Two mice in each group were injected with(−30)GFP-Cre/LNPs formulations on day 0 and 5, with 100 μg s protein foreach injection. Organs including heart, liver, spleen, lung and kidneyfrom all groups were collected 20 days after injections. The tissueswere fixed overnight in 4% paraformaldehyde (PFA) before beingsectioning into 10 μm slices. The slices were collected and stained withDAPI for fluorescence imaging.

Example 1: Preparation and Characterization of Lipid-Like Nanoparticles(LNPs)

Certain lipid-like nanoparticles (LNPs) were prepared from lipid-likecompounds of formula (I), i.e., lipidoids, by following the proceduresdescribed below. Synthesis of O17O

The following scheme was followed for synthesizing 0170.

Sodium hydride (0.72 g, 30 mmol) was added to the solution of ethyleneglycol (5.6 g, 90 mmol) in anhydrous DMF (30 mL) and stirred for 10 minat 0° C. 1-Bromotetradecane (6.0 g, 20 mmol) and KI (3.3 g, 20 mmol)were then added and the reaction mixture was kept 20 at 95° C. foranother 4 h. After cooling to room temperature, the mixture was dilutedwith cold water, extracted with ethyl acetate, and dried over anhydroussodium sulfate. Compound 1 (3.3 g, yield about 65%) was obtained aftercolumn chromatography purification on silica gel using n-hexane/ethylacetate as mobile phase. Then, compound 1 (3.3 g, 12.8 mmol) andtriethylamine (TEA, 1.9 g, 19.2 mmol) were dissolved in anhydrous 25 DCM(80 mL). Acryloyl chloride (1.4 g, 15.4 mmol) was added dropwise at 0°C., and the reaction mixture was stirred overnight. After columnchromatography purification, O17O was obtained as colorless oil (3.2 g,yield about 82%). The structure of O17O was confirmed by ¹H NMR spectrumrecorded in CDCl₃.

Synthesis of O17S

The following scheme was followed for synthesizing O17S.

To a solution of 2-mercaptoethanol (1.1 g, 14 mmol) in acetonitrile (20mL) was added 1-bromotetradecane (5.0 g, 18 mmol) and potassiumcarbonate (3.6 g, 26 mmol). The reaction solution was stirred overnightat 40° C., filtered and concentrated. Compound 2 (1.8 g, yield about48%) was obtained after column chromatography purification on silica gelusing n-hexane/ethyl acetate as mobile phase. In a manner similar tothat for the preparation of O17O, O17S was synthesized and purified asoil-like liquid (3.5 g, yield about 75%). The structure of O17S wasconfirmed by ¹H NMR spectrum recorded in CDCl₃.

Synthesis of O17Se

The following scheme was followed for synthesizing O17Se.

Potassium selenocyanate (1.5 g, 10 mmol) was added in portion to asolution of 2-bromoethanol (1.6 g, 13 mmol) in acetone (50 mL) at roomtemperature. The solution was heated to reflux for 2 h. After cooling toroom temperature, the white precipitate was filtering off and acetonewas removed by rotary evaporation under vacuum. Compound 3 was thendissolved in ethanol (25 mL) and sodium borohydride (0.9 g, 24 mmol) wasadded slowly at 0° C. After the reaction solution turned to colorless,1-bromotetradecane (4.1 g, 15 mmol) was added through a dropping funnel.The reaction was stopped by adding DI water (10 mL) after 30 min. Thenthe ethanol was removed under reduced pressure, reaction mixture wasdiluted with saturated sodium chloride aqueous solution (50 mL), andextracted with DCM (3×50 mL). Compound 5 (1.5 g, yield about 46%) wasobtained after column chromatography purification on silica gel usingn-hexane/ethyl acetate as elute. In a manner similar to that for thepreparation of O17O and O17S, O17Se was obtained as oil-like liquid (2.7g, yield about 72%). The structure of O17Se was confirmed by ¹H NMRspectrum recorded in CDCl₃.

Lipidoids Synthesis

Commercially available amine heads, e.g., Compounds 10, 17, and 63, weremixed with acrylate tails O17O, O17S, or O17Se stoichiometrically. Themixture thus obtained was stirred at 70° C. for 48 h. See FIG. 1 .Lipidoids were purified by Teledyne Isco

Chromatography system, characterized by ¹H NMR and ESI-MS, and coded asamine number (X) and O17Y (R-O17Y, Y being O, S or Se) as shown in thetable above. The typical ¹H NMR and ESI-MS spectra of 76-O17O, 76-O-17Sand 76-O17Se are shown in FIGS. 2 a and 2 b.

Lipidoid Nanoparticles Fabrication and Characterization.

Lipidoids nanoparticles (LNPs) were fabricated in sodium acetate buffer(25 mM, pH by following the simple ultrasonication and vortex proceduresdescribed above. Hydrodynamic sizes and polydispersity index (PDI) ofLNPs were measured by dynamic laser scattering (DLS) analysis. As shownin FIG. 2 c , most of the O, S and Se ethers containing LNPs had theaveraged hydrodynamic diameter (<D_(h)>) between 100-300 nm, and the PDIin the range 0.1-0.3, suitable for intracellular protein deliveryapplication. Further, as also shown in FIG. 2 c , it was found thatabout 53% of LNPs with O17O tails, about 82% of O17S LNPs, and about 65%of O17Se LNPs had <D_(h)> less than 200 nm, resulted from the effect ofincorporated chalcogen atoms on the supramolecular self-assemblybehaviors in aqueous solutions. Typical size distribution profiles of76-O17O (<D_(h)> being 170.1 nm, μ₂/

being 0.37), 76-O17S (<D_(h)> being 114.3 nm, μ₂/

being 0.24) and 76-O17Se (<D_(h)> being 129.4 nm, μ₂/Γ² 0.18) LNPs areshown in FIG. 2 d .

The morphologies of LNPs were further studied by the transmissionelectron microscopy (TEM). As shown in FIG. 3 a , spherical particleswere observed in the images of 76-O17O, 76-O17S and 76-O17Se LNPs, andthe measured number-averaged sizes (145 nm, 94 nm, and 133 nm for76-O17O, 76-O17S, and 76-O17Se, respectively) are comparable with thehydrodynamic diameters as determined by DLS. See FIG. 2 d . Themorphologies of other LNPs including 80-O17O, 80-O17S, and 80-O17Se,were also examined by their TEM images, which showed presence ofspherical particles. Subsequently, the stability of LNPs thus preparedwas examined by DLS and fluorescence measurements. As shown in FIG. 3 b, the time-dependent DLS measurements revealed that no evidentaggregation of the 76-O17O, 76-O17S, and 76-O17Se LNPs occurred duringfive days of storage under room temperature, with the relative sizechange being less than ±15%. Fluorescence resonance energy transfer(FRET) pair, DiO and DiI, loaded 76-O17Se LNPs also showed negligibleFRET ratio (I₅₇₅I(₅₇₅+I₅₀₅)) variations in five days of storage, asshown in FIG. 3 c , which indicated the structure integrity andlong-term storage stability of the LNPs.

Example 2: Evaluation of LNPs for Protein Delivery

A study was performed to evaluate the effct of LNPs prepared in EXAMPLE1 on protein delivery as follows.

In Vitro Screening of LNPs for Protein Delivery

A Cre recombinase protein fused to a negatively supercharged GFP variant((31 30)GFP-Cre) was used as a model cargo protein. The(−30)GFP-Creprotein was able to complex with cationic LNPs through electrostaticattraction and other types supramolecular interactions. The cellularuptake of LNPs could be determined by direct analysis of intracellularGFP fluorescent intensity as reported in Wang. HeLa-DsRed cells wereused in this study, which expressed red fluorescent DsRed ouponCre-mediated recombination to facilitate the functional study ofdelivered proteins in the following study.

The (−30)GFP-Cre protein loaded LNPs (GFP-Cre/LNPs) were prepared atfirst by simply mixing precalculated amount of aqueous solution of LNPsand protein at ambient conditions. For the intracellular delivery, afterincubation with GFP-Cre/LNPs nanoparticles for 6 h, the GFP-positivecells were observed using fluorescence microscopy, harvested and countedby flow cytometry. As shown in FIG. 4 a , comparing with the controlgroup, i.e., untreated HeLa-DsRed cell, bright green fluorescenceemission was observed from the GFP-Cre/Lpf2k (Lpf2k being Lipofectamine2000, a commercial transfection agent), GFP-Cre/76-O17O,GFP-Cre/76-O17S, and GFP-Cre/76-O17Se treated cells. Cells treated withthe naked protein, (−30)GFP-Cre, however, showed negligible fluorescenceemission, as compared with lipid-facilitated delivery systems, whichindicated that the naked (−30)GFP-Cre protein could not efficientlyenter into the HeLa-DsRed cells. The intracellular (−30)GFP-Cre proteindelivery efficiencies were further quantified by flow cytometry. Asshown in FIG. 4 b , both the naked (−30)GFP-Cre protein and the controlgroup showed low portions of GFP-positive cells, consistent with theresults of fluorescence microscopy shown in FIG. 4 a.

On the other hand, in the presence of LNPs, the proportions ofGFP-positive cells were increased, located in the range of 4-42%, withmost of them being around 12-18%. Delivery efficiencies of LNPs werecomparable with that of Lpf2k (about 31% of GFP-positive cells). Forinstance, the proportions of GFP-positive cells treated with(−30)GFP-Cre protein loaded 400-O17Se, 80-O17Se, and 77-O17Se LNPs werefound to be 42%, 39% and 37%, respectively.

Investigation of Structure-Activity Relationship.

The library of 51 O, S and Se ether-containing lipidoids thus preparedwas utilized to study the structure-activity relationship between LNPsand intracellular protein delivery efficacies.

More specifically, lipidoids with greater than 20% GFP-positive cellstreated with (-protein/LNP nanoparticles were defined as efficaciousLNPs (red data points in FIG. 4 b ), as compared with the bulk LNPs(black data points). The lipidoids library was then categorized intothree groups according to their hydrophobic tail structures O17O, O17S,and O17Se); each tail made up 33.3% of the library. In the efficaciousLNPs group, 21.4%, 28.6%, and 50% of lipidoids were with O17O, O17S, andO17Se tails respectively. Therefore, the relative hit rates of LNPs withO17O, O17S, and O17Se tails were -11.9%, -4.7%, and 16.7%, respectively,relative to the initial library (FIG. 4 c ). In other words, lipidoidswith O17O and O17S tails were significantly underrepresented among LNPswith delivery efficacy greater than 20%, while lipidoids with O17Se tailwas overrepresented, suggesting that O17Se tails were associated withefficacious LNPs.

It was determined that the delivery efficiencies of LNPs were related tothe chemical structures of amine heads, hydrophobic tails, substitutionnumbers and apparent pKa values. In this study, to further elucidate thestructure-activity relationship of O, S, Se ethers containing lipidoids,effects of apparent pKa value and phospholipids bilayer membranedisruption ability of the LNPs were further analyzed. Apparent pKavalues were measured following the previously reported procedures using2-(p-toluidinyl)naphthalene-6-sulphonic acid (TNS) as fluorescent probe.

The phospholipids bilayer membrane disruption ability of LNPs wasevaluated using human red blood cells (hRBCs) as model and hemoglobin asthe chromophore reporter agent. Absorbance at 405 nm (OD405) wasrecorded to assess the amount of released hemoglobin, using PBS bufferand Triton X-100 (1% v/v) as negative and positive controls,respectively, in which higher OD405 values indicate stronger membranedisruption capabilities. As shown in FIGS. 5 a and 5 b , the apparentpKa and OD405 values of LNPs were plotted against the percentages ofGFP-positive cells for each LNP, and it was found that most of theefficacious nanoparticles (with GFP-positive cells greater than 20%)were located in the regions of pKa and OD405>0.2 (gated with blue dashlines in FIGS. 5 a and 5 b ). After further examination, it was foundthat these two properties have striking effects on (−30)GFP-Cre proteintransfection efficiencies in HeLa-DsRed cells. As shown in FIG. 5 c ,when LNPs possess both of properties (i.e., pKa >5.1 and OD405>0.2), therelative hit rate to be able to mediate high transfection efficiency was77%. When one or two of the properties was removed from the LNPs, thelikelihood of achieving high transfection efficiency of(−30)GFP-Creprotein into HeLa-DsRed cells dropped significantly to8-33%. Furthermore, as to the structure-activity relationship, it wasfound that, for LNPs with O17O, O17S, and O17Se tails, the relative hitrates of above mentioned efficacy criteria were −1.9%/−14.6%,0.99%/4.2%, and 0.99%/10.5%, respectively (pKa>5.1/0D405 >0.2). See FIG.5 d . It was clear that both of the two properties were underrepresentedin the group of LNPs with O17O tails, consistent with the results shownin FIG. 4 c , in which O17O tail is was underrepresented in theefficacious lipidoid. While both properties of high pKa and OD405 valueswere overrepresented in the group of LNPs with O17S and O17Se tails.

Furthermore, according to the results shown in FIGS. 5 c and 5 d , themembrane disruption ability of these LNPs appeared to be the moreinfluential factor in determining in vitro (−30)GFP-Cre protein deliveryefficiency into HeLa-DsRed cells, as compared with the apparent pKavalues.

Example 3: (−30)GFP-Cre Protein Delivery for Gene Recombination andCytotoxicity

A study was performed to evaluate the effct of LNPs prepared in EXAMPLE1 on (−30)GFP-Creprotein delivery for gene recombination andcytotoxicity as follows.

The top 12 of LNPs identified through intracellular delivery screeningexperiments were further tested for gene recombination using HeLa-DsRedmodel cells. The expression of DsRed from Cre protein mediated generecombination was analyzed after 24 h of co-incubation with free(−30)GFP-Cre protein and protein loaded LNPs. As shown in FIG. 6 a ,naked (−30)GFP-Cre protein did not induce DsRed expression, due to itslow internalization ability, consistent with the fluorescence microscopyobservation and flow cytometry analysis demonstrated in FIG. 4 a . Mostof the test LNPs, on the other hand, efficiently delivered(−30)GFP-Creprotein and induced gene recombination, with 14-46% of thecells positive for DsRed.

More specifically, certain LNPs exhibited high protein transfectionefficiencies, namely, 76-O17S (40.8%), 76-O17Se (36.1%), 77-O17S(38.6%), 77-O17Se (31.0%), 78-O17Se (37.8%), and 80-O17S (45.6%). TheseLNPs exhibited higher or similar transfection efficiencies when comparedwith Lpf2k (33.5%).

Through MTT assay against HeLa-DsRed cells, 76-O17S, 76-O17Se, 77-O17S,and 77-O17Se LNPs showed low cytotoxicity as greater than 80% cells werealive, as compared to Lpf2k, 400-O17Se 78-O17Se, 80-O17S, and 80-O17Se,of which the cell viability was 67-77%. See FIG. 6 b.

These results indicate that 76-O17S, 76-O17Se, 77-O17S, and 77-O17Seexhibited high intracellular protein delivery and Cre-mediated genomerecombination efficacies, with lower cytotoxicity than Lpf2k.

Example 4: In vivo GFP-Cre Delivery for Gene Recombination in Ai14 Mice

A study was performed to evaluate the effct of LNPs prepared in EXAMPLE1 on GFP-Cre delivery for gene recombination in Ai14 mice as follows.

Delivering genome editing proteins in vivo has the therapeutic potentialfor treating a wide range of genetic diseases. Based on the in vitroscreening results, this study was conducted to evaluate the effect ofthe above O, S, and Se ethers containing LNPs on delivering (−30)GFP-Creprotein in vivo for Cre-mediated gene recombination.

The study used an Ai14 mouse model, which had a genetically integratedloxP-flanked STOP cassette that prevents the transcription of redfluorescent protein, tdTomato. Upon Cre mediated gene recombination, theSTOP cassette was removed, resulting in tdTomato expression. Consideringthe different performances of cargo loaded LNPs in vitro and in vivo,three LNPs with same amine heads and different tails (76-O17O, 76-O17Sand 76-O17Se) were tested in this study. Formulated LNPs(lipidoid/cholesterol/DOPE/DSPE-PEG2k=16/4/1/4, weight ratio) wereprepared. Mice were injected (intravenous injection) is with(−30)GFP-Cre loaded the formulated LNPs (GFP-Cre/76-O17O,GFP-Cre/76-0175, and GFP-Cre/76-O17Se) at day 0 and day 5 (100 μg ofprotein for each injection). Organs including heart, liver, spleen,lung, and kidney were collected at day 20 for measuring and analyzingthe tdTomato expression. As shown in FIG. 7 , under the same preparationand imaging conditions, strong tdTomato signals were observed in thesections of lung from GFP-Cre/76-0175 and GFP-Cre/76-O17Se injectedmice. Fluorescence images with lower magnification and larger field ofview were obtained. It was unexpectedly observed that the GFP/76-O17Sand GFP/76-O17Se injection induced Cre-mediated genome recombinationefficiently in the lung, as compared with the control group and thegroup treated with GFP/76-O17O. Therefore, a composition containing LNPsof this invention is useful for lung disease treatment.

Notably, both the in vitro screening results and the in vivo testsshowed that lipidoids with same amine heads and different hydrophobictails possessed very different physicochemical properties, intracellulardelivery efficacies, and genome recombination profiles.

Example 5: Delivery of Cas9:sgRNA RNP for Genome Modification

A study was performed to evaluate the effct of LNPs prepared in EXAMPLE1 on the delivery of Cas9:sgRNA RNP for genome modification as follows.

The Cas9:sgRNA RNP targeting genomic GFP reporter gene and GFP-HEK cellswere used in this study. The morphologies of Cas9:sgRNA loaded LNPs wereexamined by TEM, and typical image of Cas9:sgRNA loaded 76-O17Se LNP(Cas9:sgRNA/76-O17Se) was obtained. For the intracellular delivery,GFP-HEK cells were harvested after treating with Cas9:sgRNA/LNPsnanocomplexes for 48 h. GFP gene knockout efficacy was further evaluatedby flow cytometry. As shown in FIG. 8 a , naked Cas9:sgRNA RNP did notio induce GFP gene knockout, while the knockout efficiency ofCas9:sgRNA/Lpf2k was relatively high, with 63% of GFP-negative cells.When using O, S, and Se ethe-containing LNPs as delivery vehicles, theGFP-HEK cells showed a loss of 14%-58% GFP expression. In particular,50.2%, 57.7%, 54.7% and 57.4% of GFP knockout were observed when cellswere treated with Cas9:sgRNA loaded with 76-O17Se, 80-O17Se, 81-O17Se,and 400-O17Se LNPs. These lipidoids could efficiently deliver genomeediting proteins into different mammalian cell lines in vitro, based onthe results of gene recombination of Cre protein in HeLa-DsRed cells andGFP gene knockout of Cas9:sgRNA RNP delivery in GFP-HEK cells.

In vitro cytotoxicity of Cas9:sgRNA/LNPs against GFP-HEK cells was alsoevaluated by the MTT assay. As shown in FIG. 8 b , the cell viabilitieswere determined to be 67%-119% after incubation with Cas9:sgRNA/LNPs at37° C. for 48 h, indicating that the certain LNPs were non-cytotoxic toGFP-HEK cells, while some showing cell viability the same as that ofLpf2k (cell viability about 66%) under the same experimental conditions.It was also observed that two LNPs with high Cas9:sgRNA deliveryefficiencies, i.e. 80-O17Se and 400-O17Se, exhibited cell viabilitysimilar to that for Lpf2k, namely, 68.2% and 66.7% of cell viability for80-O17Se and 400-O17Se, respectively. Unexpectedly, 76-O17Se and81-O17Se LNPs showed both high Cas9:sgRNA transfection efficiency (50.2%and 54.7%) and low cytotoxicity (76.3% and 97.7% of cell viability after48 h of incubation).

These results indicate that LNPs formed from lipid-like compounds offormula (I) exhibited high protein transfection efficiency and lowcytotoxicity.

Methods and Materials Preparation of Blank and Cargo-Loaded LipidoidNanoparticles

Lipidoids were fabricated into nanoparticles for all deliveryapplications. Briefly, lipidoids were mixed with sodium acetate buffer(25 mM, pH 5.2), sonicated for 30 mM in an ultrasonic bath, followed byanother 30 min of vigorous vortexing. The as-prepared blank LNPs werestored at 4° C. For Cy5-RNA/LNP, mRNA/LNP and protein/LNP complexation,blank lipidoid nanoparticles were mixed with RNA molecules or(−30)GFP-Cre protein in PBS buffer (pH 7.4) following our previouslyreported procedures and incubated at room temperature for another 30 mMbefore use. Typical procedures for Nile red encapsulation are asfollows: 5 μL of Nile red stock solution in acetone was added into anempty vial, which was then placed in a vacuum oven to completely removethe organic solvent. Then, a predetermined amount of blank LNP stocksolution (1.0 mg/mL) was added into the vial. The mixture was sonicatedfor 40 mM in an ultrasonic bath and stirred overnight at roomtemperature. The final concentration of Nile red was adjusted to6.6×10-7 mol L-1 and 6×10-7 mol L-1 for thiol triggered release studyand cell incubation, respectively, by diluting with PBS as necessary.Typical procedures for CPT and DiO/DiI FRET pair encapsulation are asfollows: 100 μL of DiO/DiI stock solution in Me0H was charged into anempty vial and placed in a vacuum oven to remove the organic solvent.Lipidoids (2.0 mg) in 200 μL of methanol were then added into the vialand stirred to produce a homogeneous solution. Then, 600 μL of DI waterwas added dropwise in 10 mM with continous stirring. The resultingmixture was dialyzed against DI water for 24 h (Thermo ScientificSlide-A-Lyzer Dialysis Cassette, MWCO=3500 Da), and fresh water wasreplaced every 4 h. Typical procedures for encapsulation of calcein anddoxonorubicin hydrochloride are as follows: precalculated amounts ofcalcein or Dox stock solutions in DI water was diluted into 800 μL withsodium acetate buffer, and used as the selective solvents to trigger theself-assembly process of lipidoids in methanol (5 mg/mL), respectively.The unloaded calcein or Dox was removed by dialysis against DI water(Thermo Scientific Slide-A-Lyzer Dialysis Cassette, MWCO=3500 Da).

Intracellular Delivery of Cargo-Loaded Lipidoid Nanoparticles

For the intracellular uptake study, HeLa or HeLa-DsRed cells were seededin 48-well plate with an initial seeding density of 2×104 cell/well.After 24 h of incubation at 37° C., 5% CO2, NR or (−30)GFP-Cre loadednanoparticles were added to the cells and incubated for certain time(1-8 h) before fluorescence microscopy (BZ-X Analyzer) observation andflow cytometry (BD FACS Calibur, BD Science, CA) analysis (redfluorescence emission from NR and green fluorescence emission from GFP).The final concentration of NR is 6×10-7 mol L-1. The final concentrationof (−30)GFP-Cre protein concentration is 25-100×10-9 mol L-1. For smallmolecular anticancer drugs delivery, HeLa cells were seeded in 96-wellplate with an initial seeding density of 5×103 cell/well. After 24 h ofincubation at 37° C., 5% CO2, Dox, CPT or Oxa loaded nanoparticles wereadded to the cells and incubated for 8 h followed by media change. Thecells were then incubated for another 40 h before cell viabilityanalysis. For mRNA delivery, HeLa, B 16F10, HEK 293, NIH 3T3 or Jurkatcells were seeded in 48-well plate with an initial seeding density of2×104 cell/well. After 24 h of incubation at 37° C, 5% CO2, mRNA loadednanoparticles were added to the cells and io incubated for another 24 hbefore fluorescence microscopy and flow cytometry analysis. For proteindelivery, HeLa-DsRed cells were seeded in 48-well plate with an initialseeding density of 2×104 cell/well. After 24 h of incubation at 37° C,5% CO2, (−30)GFP-Cre protein loaded nanoparticles were added to thecells and incubated for 8 h followed by a complete media change. Thecells were then incubated for another 16 h (24 h of incubation in total)before fluorescence microscopy and flow cytometry analysis.

In Vitro and in Vivo Toxicity Assay

Cell viabilities of HeLa and HeLa-DsRed were measured using the standardMTT assay. In a 96-well plate, after incubating HeLa or HeLa-DsRed cellswith blank or cargo-loaded nanoparticles, the MTT reagent (5 mg/mL, in30 μL PBS buffer) was added and the cells were incubated for another 4 hat 37° C. The cell culture media was then carefully removed and 200 μLof DMSO was added to each well. The DMSO solution was then transferredinto a clean 96-well plate and the absorbance at 570 nm was recorded bya microplate reader. All experiments were performed in quadruplicate.

For in vivo toxicity studies, the body weights of untreated andnanoparticles injected Balb/c mice (housed in a temperature and humiditycontrolled facility with a 12 h light/dark cycle) were measured at day1, 3, 5, 7, 9, 11, 13 and 14. Biological functions of kidney and liverwere examined by the serum biochemical tests and concentrations ofcreatinine, urea, aspartate aminotransferase (AST), and alanineaminotransferase (ALT) were measured using corresponding detection kits(MilliporeSigma) following manufacturers' protocols.

In Vivo Protein and mRNA Delivery to Ai14 Mouse

Similar to the in vitro transfection study, lipidoid nanoparticles wereprepared for mRNA or protein loading and in vivo delivery. Ai14 micewere housed in a temperature and humidity controlled facility with a 12h light/dark cycle. Three mice in each group were injected(intravenously or intramuscularly) with Cre mRNA-loaded or (−30)GFP-Creprotein-loaded LNPs formulations. Organs including heart, liver, spleen,lung and kidney from all groups were collected at day 10 (intramuscularinjection) or 14 (intravenous injection)after injection. The tissueswerefixed overnight in 4% paraformaldehyde (PFA) and dehydrated in 30%sucrosebefore beingfrozein in OCT and sectioned into 10-15 μmslices.Thesliceswere then collected and stained with DAPI for fluorescenceimaging(BZ-X Analyzerfluorescence microscopy).

Example 6: Cholesteryl Lipidoid Synthesis, Nanoparticles Fabrication andCharacterization

Synthesis of Py-SS-Chol

Cholesteryl chloroformate (10.71 g, 23.85 mmol) was dissolved inanhydrous DCM (50 mL) and added into the DCM solution of Py-SS-NH2 (4.47g, 23.99 mmol) and TEA (3.71 g, 36.69 mmol) dropwise at 0° C. Thereaction mixture was stirred overnight and Py-is SS-Chol was obtained asslightly yellow viscous solid (4.89 g, yield˜34.26%) after silica gelcolumn chromatography purification using ethyl acetate, dichloromethaneand n-hexane as the mobile phase.

Synthesis of OH-SS-Chol

Py-SS-Chol (3.55 g, 5.93 mmol) and acetic acid (600 μL) were dissolvedin DCM (100 mL). 2-Mecaptoethanol (0.51 g, 6.52 mmol) was then addeddropwise, and the reaction mixture was maintained at 35° C for another24 h with continuous stirring. OH-SS-Chol was purified by silica gelcolumn chromatography using ethyl acetate and n-hexane as mobile phaseand a colorless solid was obtained (2.74 g, yield˜81.63%).

Synthesis of OChoiB

OH-SS-Chol (2.41 g, 4.26 mmol) and TEA (0.65 g, 6.39 mmol) weredissolved in anhydrous DCM (100 mL). Acryloyl chloride (0.46 g, 5.11mmol) was added dropwise at 0 oC. The reaction mixture was stirredovernight and OCholB was obtained as a colorless solid (2.52 g, yield˜95.68%) after silica gel column chromatography purification using ethylacetate, dichloromethane and n-hexane as mobile phase.

Synthesis of lipidoids

The cholesterol-containing acrylates tails shown above were reacted withhead amines R_(a)-NH₂ (i.e., Compounds 75-78, 80, 81, 87, 90, and 304.)to afford the following lipid-like compounds:

Preparation of Blank and Cargo-Loaded Lipidoid Nanoparticles

Lipidoids were fabricated into nanoparticles for all deliveryapplications. As shown in FIG. 10B, most nanoparticles showed averagediameters in the range of 70-300 nm and PDI 0.1-0.3. The sizes of LNPsself-assembled from lipidoids with OCholB tails are similar to ourpreviously reported LNP libraries with alkyl chains. The relative lowPDI values indicated the uniformity of these nanoparticles.

The morphologies of OCholB fully substituted LNPs were then examined bytransmission electron microscopy (TEM). As shown in FIG. 10C, sphericalvesicle-like structures, which are hollow spheres with hydrophobicbilayer walls sandwiched by hydrophilic internal and external coronas,were observed from 75-OCholB, 76-OCholB, 77-OCholB, 78-OCholB,80-OCholB, 81-OCholB, and 304-OCholB. In contrast, the vesicularstructures were not well-formed for 87-OCholB and 90-OCholB compared totheir counterparts, and amorphous aggregates were observed instead. TEMimaging revealed the largest particles to be 81-OCholB (216.9 nm) and304-OCholB (394.0 nm), which is consistent with the DLS measurementresults as shown in FIG. 10B.

The cytotoxicity of OCholB fully substituted LNPs (75-OCholB, 76-OCholBand 77-OCholB) and 016B LNPs (75-016B, 76-016B and 77-016B) were testedside-by-side under different conditions, i.e., low dosage/short exposuretime and higher dosage/long exposure time, against HeLa cell line usingthe standard MTT assay. As shown in FIG. 10E, at the low dosage/shortexposure time conditions (i.e., [lipidoid]=1.0 or 3.3 μg mL⁻¹, exposuretime=8 h), all the OCholB and 016B LNPs showed negligible cytotoxicity,with cell viability of >83% reported for all lipidoids (e.g. when[lipidoid]=3.3 μg mL⁻¹, the viabilities of 75-OCholB and 75-016B treatedcells are 86.5% and 87.7%, respectively). When the dosage and exposureduration were both increased (i.e., [lipidoid]=47 or 91 μg mL⁻¹,exposure time=24 h), OCholB fully substituted LNPs treated cells showedsignificantly higher viabilities comparing to those treated with 016BLNPs (when [lipidoid]=47 μg mL⁻¹, the cell viabilities are75-OCholB/75-016B=57.4%/41.9%, 76-OCholB/76-016B=65.5%/32.8%,77-OCholB/77-016B=79.6%/29.0%; when [lipidoid]=91 μg mL⁻¹, the cellviabilities are 75-OCholB/75-016B=55.1%/29.5%,76-OCholB/76-016B=60.2%/24.3%, 77-OCholB/77-016B=64.1%/21.9%). Theseresults show that the cholesteryl lipidoids have lower cytotoxicitycomparing with the lipidoids with the linear alky chain. Furthermore, wecompared the biocompatibility of our newly developed OCholB LNPs againstthat of the commercially available and widely used cationic transfectionagent, lipofectamine 2000 (Lpf2k). Lpf2k is shown to be highly efficientfor both protein and nucleic acids delivery; however its cytotoxicity isoften a major concern, especially when the targeted cells are exposed toa relative high dosage and long incubation duration. As shown in FIG.10E, when the HeLa cells were treated with 47 and 91 μg mL⁻¹ of Lpf2kfor 24 h, their viabilities were determined to be 8.9% and 6.8%, whichare much lower than that of OCholB LNPs treated cells under the sameconditions. Above all, the in vitro cytotoxicity tests demonstrated theexcellent biocompatibility of the newly developed cholesteryl-containing(OCholB) LNPs.

Example 7: Thiol-Responsiveness, Loading and Triggered Release of GuestMolecules

The thiol-trigged degradation and dissociation of the OCholB LNPs werestudied by time-dependent DLS measurements and TEM observation.Typically, as shown in FIG. 11A, in the presence of 10 mM of1,4-dithiothreitol (DTT), which has been widely used in io previousstudies for mimicking intracellular reductive conditions,' we observedthe increase of the relative sizes of 75-OCholB, 76-OCholB and 77-OCholBgradually increased along incubation duration, as 566.4%, 498.5% and1591.4% respectively, in first 2 h. Nanoparticle size was then typicallymaintained over the following 4 h, with the exception of 75-OCholB,which showed 1315.7% increase in size at 6 h. The typical morphologiesof DTT treated LNPs were then examined by TEM and images are shown inFIG. 11B. The absence of well-formed vesicular structures as shown inFIG. 10C and large aggregates at micrometer scales (which are consistentwith the results obtained from DLS measurements; FIG. 11A) withamorphous structures were observed for 75-OCholB, 76-OCholB and77-OCholB LNPs. The disruption of the vesicle structure is considered tobe resulted from the thiol-exchange and disulfide bond cleavagereactions of OCholB lipidoids. We next investigated whether thethiol-containing molecules (e.g. albumin, cysteine, homocysteine,cysteinylglycine, etc.) in the serum may induce the structuraldisintegration of these disulfide bond-containing LNPs. The stabilitiesof LNPs in the presence of 20 μM of L-cysteine (Cys), which mimics thefree thiols on the small- and macromolecules presented in the serum,were examined Shown in FIG. 11A, we observed <25% changes inhydrodynamic diameter at any of the 1 h intervals over the span of thisstudy for all the three tested LNPs, and in fact 12.2%, 14.6% and 7.2%decreases in size were observed for 75-OCholB, 76-OCholB and 77-OCholBafter 6 h of incubation, respectively. The size changes ofcysteine-treated LNPs showed negligible difference when compared withthe untreated control groups (FIG. 11A), indicating the good stabilityof OCholB LNPs under the conditions mimicking the concentration of freethiols in the blood serum. Furthermore, the relative size variations ofthe OCholB LNPs after 24 h of incubation in the presence of either 10 mMDTT or 20 μM Cys were determined and the results are shown in FIG. 11C.With 10 mM of DTT and after 24 h of treatment, 75-OCholB, 78-OCholB and304-OCholB LNPs showed the greatest size changes, with 2872.4%, 4642.8%and 3849.6% increase in averaged hydrodynamic diameters observed; amoderate size increase of 766.9%-1266.4% were recorded for 76-0Cho1B,77-0Cho1B, 80-OCho1B and 81-0Cho1B LNPs; while both 87-OCholB (210.3%)and 90-OCholB (179.5%), which were unable to form consistent vesiclesbased on the TEM images (FIG. 10C), showed a minimal size increase over24 hours. On the other hand, all LNPs incubated with 20 μM Cysteine for24 h showed minimal size changes, similar to the untreated controlgroups (FIG. 11C). These results demonstrated the kinetics of thedegradation of these OCholB LNPs in relation to intracellular andextracellular reducible environments. While the degrees of size changesvary between lipidoids with different amine head groups, all lipidoidswere more responsive to DTT (modeling intracellular conditions) than toCysteine (modeling conditions in the blood serum). Furthermore, theOCholB LNPs showed relative good stability in the presence of lowconcentrations of thiols (mimicked by 20 μM Cysteine treatment) whichindicates that these new LNPs could be used for systemic drug delivery.

Example 8: Drug Encapsulation using OCholB LNPs

Next, the capabilities of OCholB LNPs as nanocarriers to encapsulatecargo molecules with various physical properties were studied. In thiscontext, coumarin (Excitation (Ex.) 350 nm, Emission (Em.) 448 nm) andNile red (NR; Ex. 520 nm, Em. 613 nm) as representative small molecularhydrophobic cargoes, calcein (Ex. 475 nm, Em. 529 nm) as arepresentative small molecular hydrophilic cargo, and (−30)GFP-Crerecombinant fluorescent protein (Ex. 420 nm, Em. 510 nm) and doublestranded, Cy5 labeled RNA (Cy5-RNA, 13kDa; Ex. 625 nm, Em. 672 nm) asrepresentative macromolecular hydrophilic cargoes were used as the modelcargoes. 75-OCholB was chosen as the model lipid carrier in the study.FIG. 11D showed that the all the cargoes can be successfully loaded intothe 75-OCholB LNPs. The cargo molecules were loaded into LNPs eitherthrough hydrophobic interactions (coumarin and NR), electrostaticinteractions (calcein, (−30)GFP-Cre and Cy5-RNA), or physicalencapsulation. Furthermore, as shown in FIG. 3D, simultaneousencapsulation of cargo molecules could be also achieved using thehydrophobic fluorescence resonance energy transfer (FRET) pair, DiO andDiI (Ex. 425 nm, Em. 504 nm (DiO) and 578 nm (DiI)), as a model, whichdemonstrates the possibility of using these OCholB LNPs to load multipletypes bioactive molecules simultaneously for combination therapies. ³⁰By taking advantages of the supramolecular interactions (e.g.electrostatic interaction and hydrogen bonding) and/or encapsulatingcargoes during the self-assembly process, both small and macromolecularhydrophilic molecules (e.g. genome editing platforms and cell signalinhibitors) could be readily loaded and delivered by the newly developedLNPs.

The reduction triggered release behavior of encapsulated cargoes wasthen studied by using nile red loaded 75-OCholB LNPs (NR/75-OCholB),taking advantage of the microenvironmental polarity sensitivephotophysical property of nile red. As shown in FIG. 11E, in thepresence of 1 mM, 5 mM and 10 mM of DTT, 33.5%, 61.7% and 67.4% ofencapsulated nile red were released from nile red/75-OCholB LNPs within2 h, and 44.0%, 84.2% and 86.4% were released in 6 h of incubation,respectively. In the meantime, 20 μM Cysteine treated NR/75-OCholBreleased 4.2% of nile red in 2 h and 6.9% in 6 h, which could beascribed to the previously discussed minimal structural andmorphological changes of the LNPs under the stimulus of lowconcentration of thiols (FIG. 11A and 11C). The triggered release ofhydrophilic fluorescent dye, calcein, which possesses a self-quenchingfeature at high concentrations, was further studied. 31 The fluorescenceintensities of DTT (1 mM, 5 mM and 10 mM) treated calcein/75-OCholB LNPsincreased 4.7-6.4 folds after 12 h incubation comparing to untreatedcontrol LNPs and 20 NM of Cysteine treated groups (FIG. 11F).Furthermore, the binding affinity of OCholB LNPs with negatively chargedmacromolecular cargo, Cy5-RNA, was examined. It was found that at a 10/1weight ratio (lipidoid/Cy5-RNA), 82.9% of the RNA molecules could beefficiently complexed with 75-OCholB LNPs, while the binding efficacydramatically reduced to 15.5% when DTT (10 mM, 24 h) treatednanoparticles were used (as 84.5% unbound Cy5-RNA was determined; FIG.11G). Additionally, similar to the responsiveness study, it isreasonable that the cargo release profiles could depend on both of thespecies and concentrations of the thiols-containing regents. Above all,OCholB LNPs loaded with cargoes are relatively stable in the presence oflow concentration of thiols and the triggered release behaviors of cargomolecules with different physicochemical properties(hydrophobic/hydrophilic, low/high molecular weight, etc.) could beexpected.

Example 9: Internalization Studies

The cell (HeLa and HeLa-DsRed cell lines) internalization studies wereconducted using small molecular hydrophobic (nile red) and hydrophilic(calcein) fluorescent dyes and macromolecular fluorescent recombinantprotein ((−30)GFP-Cre) loaded OCholB LNPs. The stabilities ofcargo-loaded LNPs were examined at first using time-dependent DLS andfluorescence measurements. As shown in FIG. 12A, the fluorescenceintensity of FRET pair DiO and DiI encapsulated LNPs (DiO-DiI/75-OCholB,DiO-DiI/76-OCholB and DiO-Di1/77-OCholB) showed negligible variations onFRET ratio (I₅₇₅/I₅₇₅+I₅₀₅) after 7 days of storage.

The internalization kinetics and efficiencies of OCholB LNPs were thenstudied using NR loaded LNPs and HeLa cells. As shown in FIG. 12B,comparing to the untreated control cells, all the cells incubated withNR/LNPs (NR/75-OCholB, NR/76-OCholB and NR/77-OCholB) showed thepercentage of NR positive (NR³⁰) cells gradually increasing over time,which means the internalization process of NR/LNPs is exposure timedependent over the time scale of this study. Furthermore, bothNR/75-OCholB and NR/77-OCholB showed a similar NR⁺ population growthpattern and decreased growth rate after 4 h of exposure (at 4 h , theNR⁺ percentages for NR/75-OCholB, NR/76-OCholB and NR/77-OCholB treatedcells are 85.2%, 65.0% and 90.3%, respectively); while in general,NR/76-OCholB showed a constant increase rate and a lower NR⁺ percentageafter 8 h comparing to NR/75-OCholB and NR/77-OCholB. Next, all of theNR delivery efficiencies of eight OCholB LNPs were determined after 8 hof exposure. As shown in FIG. 12C, NR/75-OCholB, NR/76-OCholB, isNR/77-OCholB, NR/78-OCholB and NR/80-OCholB showed highest deliveryefficiencies, with 85.2%, 65.0%, 90.3%, 92.3% and 71.4% of cellsdetermined as NR±; 27.0%, 12.9% and 22.8% NR⁺ cells were recorded forNR/81-OCholB, NR/90-OCholB and NR/304-OCholB; NR/87-OCholB showed lowesttransfection efficacy, comparable to the untreated control group, whichmeans 87-OCholB cannot efficiently deliver NR into HeLa cells under thetested conditions. Representative fluorescent images of NR/LNPs(NR/75-OCholB, NR/76-OCholB and NR/77-OCholB) treated HeLa cells areshown in FIG. 12D, from which it is easily observed that NR wasdelivered into the cells and mainly distributed in cytoplasm, by OCholBLNPs. Next, the internalization of negatively charged hydrophilicfluorescent dye, calcein, was studied. As shown in FIG. 12E, after 8 hof exposure, the free calcein molecules cannot enter into the cellsefficiently, which is consistent with previously reported results.Calcein/75-OCholB treated cells showed relatively high green fluorescentintensity, as —9.4 folds higher mean fluorescent intensity was recordedwhen compared to the free calcein-treated cells. These results provedthat the OCholB LNP may serve as efficient nanocarriers forintracellular delivery of both hydrophilic and hydrophobic cargomolecules. Next, the use of OCholB LNPs for the intracellular deliveryof macromolecular cargo was explored, using fluorescent recombinant(−30)GFP-Cre protein and HeLa-DsRed cells as the model system. As shownin FIG. 12F, naked (−30)GFP-Cre and (−30)GFP-Cre loaded Lpf2k((−30)GFP-Cre/Lpf2k) were used as negative and positive controls, as ithas been demonstrated that the naked (−30)GFP-Cre protein cannot enterinto the cells and Lpf2k is highly efficient for (−30)GFP-Cre delivery.Delivery efficiency was tested at a range of (−30)GFP-Cre proteinconcentrations (25, 50 and 100 nM), with a consistent lipid/proteinratio (i.e., delivery with the final lipid concentration of 1.7, 3.3,and 6.6 μg mL⁻¹ respectively). 33.2%, 43.8% and 74% of GFP positive(GFP±) cells were recorded for (−30)GFP-Cre/Lpf2k treated cells with theconcentration of (−30)GFP-Cre 25 nM, 50 nM and 100 nM, respectively. Atthe 25 nM (−30)GFP-Cre concentration, (−30)GFP-Cre/76-OCholB (66.4%),(−30)GFP-Cre/77-OCholB (66.4%), (−30)GFP-Cre/80-OCholB (54.7%), and(−30)GFP-Cre/81-OCholB (40.9%) all showed higher transfectionefficiencies than (−30)GFP-Cre/Lpf2k. At the higher (−30)GFP-Creconcentrations (i.e. 50 nM and 100 nM), (−30)GFP-Cre/75-OCholB (42.3%and 93.1% GFP⁺ cells with 50 nM and 100 nM respectively),(−30)GFP-Cre/76-OCholB (96.3% and 98.7%), (−30)GFP-Cre/77-OCholB (90.7%and 97.9%), (−30)GFP-Cre/78-OCholB (46.1% and 90.1%),(−30)GFP-Cre/80-OCholB (88.9% and 97.0%), and (−30)GFP-Cre/81-OCholB(87.4% and 91.5%) showed comparable or even higher transfectionefficacies than Lpf2k. 48.8% of GFP⁺ cells were obtained from(−30)GFP-Cre/304-OCholB treated cells with the concentration of protein100 nM, while both (−30)GFP-Cre loaded 87-OCholB and 90-OCholB showedlowest delivery efficiencies comparing to other OCholB LNPs. This resultis consistent with the small molecular NR delivery results as shown inFIG. 12C, indicating these two nanoparticles are probably inefficientfor intracellular delivery applications. Although many of the OCholBLNPs as well as Lpf2k possessed similar GFP⁺ cells percentages (FIG.4F), as shown in FIG. 12G ([lipidoid]=6.6 μg mL⁻¹ and R-30)GFP-Crel=100nM), further analysis revealed that the mean fluorescence intensities of(−30)GFP-Cre loaded nanoparticles treated cells varied significantly,indicating some of the LNPs (75-OCholB, 76-OCholB, 77-OCholB, 80-OCholBand 81-OCholB) are much more efficient than others (Lpf2k and78-OCholB), as higher mean fluorescence intensities represent largeramount of (−30)GFP-Cre proteins were successfully delivered into thecells. The typical flow cytometry profiles of naked (−30)GFP-Cre proteinand (−30)GFP-Cre loaded nanoparticles ((−30)GFP-Cre/75-OCholB,(−30)GFP-Cre/76-OCholB, (−30)GFP-Cre/77-OCholB and (−30)GFP-Cre/Lpf2k)treated HeLa-DsRed cells were shown in and FIG. 12H ([lipidoid]=6.6 μgmL⁻¹ and R-30)GFP-Crel=100 nM), which is consistent with the statisticalresults shown in FIG. 12G. Furthermore, the representative fluorescentimages from the protein and protein/nanoparticle treated HeLa-DsRedcells are also shown in FIG. 121 . Strong green fluorescence signalsfrom (−30)GFP-Cre/75-OCholB, (−30)GFP-Cre/76-OCholB,(−30)GFP-Cre/77-OCholB and (−30)GFP-Cre/Lpf2k and negligible signalsfrom (−30)GFP-Cre treated- and untreated cells were detected, which isalso consistent with the results from flow cytometry analysis. FIG. 12Jshows typical bight field images of (−30)GFP-Cre/LNPs((−30)GFP-Cre/75-OCholB, (−30)GFP-Cre/76-OCholB and(−30)GFP-Cre/77-OCholB) treated cells ([lipidoid]=6.6 μg mL⁻¹ andR-30)GFP-Crel=100 nM) and no evident morphological change were observedcomparing to the untreated cells, which further demonstrate thebiocompatibility of the OCholB LNPs. Taken together, this data indicatesthat most of the newly developed fully substituted OCholB LNPs areefficient for the delivery of small molecular hydrophobic andhydrophilic cargoes as well as macromolecular cargoes into mammaliancells in vitro.

Example 10: Intracellular Delivery of Small Molecular Anticancer Drugs

The possibility of using OCholB LNPs to deliver both hydrophobic andhydrophilic small molecular drugs was explored. Doxorubicinhydrochloride (Dox) (water soluble), and camptothecin (CPT) andoxaliplatin (Oxa) (water insoluble) were encapsulated into LNPs (seeexperimental section) and tested against HeLa cells. The successfulencapsulation of small molecular drugs was demonstrated by examining theabsorption and fluorescence emission spectra of Dox (Ex. 495 nm, Em. 594nm) and CPT (Ex. 360 nm, Em. 446 nm) loaded 75-OCholB LNP, in which thecharacteristic absorbance and emission peaks of Dox and CPT wereobserved, as shown in FIG. 13A. Using corresponding standard curves, thedrug loading contents(DLC%=[_(loaded drug)]/[W_(loaded drug)+W_(lipidoid)]100%) weredetermined to be 19.2% and 5.2% for Dox and CPT, respectively. Then theinternalization of Dox loaded 75-OCholB LNP (Dox/75-OCholB) was studiedafter 8 h of exposure using flow cytometry. As shown in FIG. 13B, insharp contrast to calcein (FIG. 12E) and (−30)GFP-Cre (FIG. 4 f ), freeDox could be readily internalized by HeLa cells after 8 h of incubation.Dox/75-OCholB treated HeLa cells also showed a comparable meanfluorescence intensity as free Dox treated cells, and are ˜28.9 foldshigher comparing to the untreated control cells, indicating theDox/75-OCholB nanoparticles could be efficient for intracellulardelivery of Dox under this condition.

Dose-dependent cytotoxicity was then examined From FIG. 13C, theDox/75-OCholB showed a similar concentration-dependent cytotoxicityprofile as free Dox against HeLa cells (8 h of exposure; MTT assay after48 h of incubation). Meanwhile, blank 75-OCholB LNPs showed negligibletoxicity under the same conditions. The cell viabilities treated byblank LNPs maintained to be >80%, which further validates the safety ofthe OCholB LNPs. Next, hydrophobic anticancer drugs, CPT and Oxa wereencapsulated into LNPs (CPT/75-OCholB and Oxa/75-OCholB) and the cellviabilities of CPT/75-OCholB and Oxa/75-OCholB treated HeLa cells (bothwith 8 h of exposure; [CPT]=1.8 μg mL⁻¹; [Oxa]=2.4 μg mL⁻¹) after 48 hof incubation were determined as 42.4% and 66.9% (FIG. 13D). Overall,the Dox and Oxa encapsulated 75-OCholB LNPs showed comparable or evenhigher toxicities than their free counterparts; while CPT loaded75-OCholB was less efficient than the free CPT. This indicates that thephysicochemical properties of the cargo drugs could have a huge impacton the delivery performances of OCholB LNPs, which in principle may alsobe true for other carrier systems.

Example 11: Intracellular Delivery of mRNA

Messenger RNA delivery has great potentials for cancer therapy, proteinreplacement therapy and neurological disorder treatments. 42 Theintracellular delivery of mRNA using OCholB LNPs was studied using GFPmRNA and different cell lines (HeLa, B 16F10, HEK-293, NIH/3T3 andJurkat). The weight ratio of LNP/mRNA was optimized at first using HeLacells. As shown in FIG. 14A, by fixing the final concentration of mRNAas 0.86 μg mL⁻¹ and increasing the LNP/mRNA weight ratio from 0/1 (i.e.free mRNA, without LNP) to minimal GFP⁺ cells were determined after 24 hof exposure when the LNP/mRNA ratio is less than 1/1 (0.7%, 0.8% and1.4% of GFP⁺ cells were determined for LNP/mRNA=0/1, 0.5/1 and 1/1,respectively). Gradual increase on GFP⁺ populations were observed whenthe ratio is increased from 2/1 to 15/1, and 24.3%, 39.2%, 64.7% and68.9% of GFP⁺ cells were recorded for LNP/mRNA ratios of 2/1, 5/1, 10/1and 15/1. The weight ratio of LNP/mRNA=was then chose for the followingmRNA delivery studies. The intracellular delivery of GFP mRNA was alsofound to be dose dependent in the mRNA concentration range of 0.027-1.5μg mL⁻¹, as continuous increase in GFP⁺ cells (from 3.3% to 76.3%) wereobserved when increasing the dosage of mRNA/LNPs (FIG. 14B). Then theintracellular delivery efficiencies of all fully substituted OCholB LNPswere tested against HeLa cells, and Lpf2k and naked GFP mRNA were usedas controls (LNP/mRNA=10/1; [mRNA]=0.86 μg mL⁻¹; 24 h exposure). Asshown in FIG. 14D, naked mRNA induced neglectable GFP⁺ cells, which iscomparable to the untreated cells, while Lpf2k is highly efficient formRNA delivery, with 98.1% GFP⁺ HeLa cells. As to the OCholB LNPs,75-OCholB (62.0% of GFP⁺ cells), 76-OCholB (79.8%), 77-OCholB (73.5%),78-OCholB (67.1%), 80-OCholB (55.6%), 81-OCholB (51.6%) and 304-OCholB(52.1%) are all determined to be effective. Typical fluorescent imagesof mRNA/LNPs (mRNA/75-OCholB, mRNA/76-OCholB and mRNA/77-OCholB) treatedHeLa cells are shown in FIG. 14C. Comparing to untreated HeLa cells,strong green fluorescent signals were recoded from nanoparticlesincubated cells, which is consistent with the flow cytometry data asshown in FIG. 14D. Meanwhile, both 87-OCho1B (6.1%) and 90-OCholB (2.5%)are found to be inefficient for GFP mRNA delivery into HeLa cells, whichis consistent with the internalization studies using NR (FIG. 12C) and(−30)GFP-Cre protein (FIG. 12F) as the fluorescent reporters. It wasrevealed that consistency of the delivery performances may exist amongthese OCholB LNPs, and the internalization efficacies of non-active LNPsstayed minimal regardless of the properties of loaded cargoes.

To examine the delivery spectrum of the newly developed LNPs, the GFPmRNA loaded OCholB LNPs were then challenged against other four types ofcell lines. B 16F10 io (mouse melanoma cells), HEK 293 (human embryonickidney cells), NIH 3T3 (mouse embryonic fibroblast cells) and Jurkat(human T lymphocyte cells) cells were tested (LNP/mRNA=10/1; [mRNA]=0.86μg mL⁻¹; 24 h exposure) and the results are shown in FIG. 14D. As thepositive control, Lpf2k turned out to be very efficient to deliver GFPmRNA into B 16F10 (63.2% of GFP⁺ cells), HEK 293 (80.1%) and NIH 3T3(70.1%) cells, while is slightly less efficient to Jurkat cell (28.7%),which is usually considered to be one of the most difficult-to-transfectcell lines. As to the OCholB LNPs, in general, 87-OCholB, 90-OCholB and304-OCholB were proved to be less efficient to deliver mRNA into allthese cell lines; while other six OCholB LNPs were found to be much moreefficient. For example, 63.0%, 54.1% and 50.1% of GFP⁺ cells weredetermined from mRNA/75-OCholB treated Bl6F10, HEK 293, NIH 3T3 andJurkat cells; and the numbers for mRNA/76-OCholB treated cells were70.7%, 75.8%, 24.1% and 7.7%, respectively. It was obvious that not onlythe types of lipidoids, but also the target cell lines could have hugeimpacts on the delivery efficacy. Overall, the positive control, Lpf2k,is a relatively high-activity broad-spectrum transfection reagent; mostof the OCholB LNPs (6 out of 9) are also effective broad-spectrumtransfection reagents under the tested conditions. Some of the OCholBLNPs (e.g. 75-OCholB, 77-OCholB and 78-OCholB) showed particularadvantages over Lpf2k regarding the efficacy of delivery to Jurkatcells. It was expected that through further formulation optimization,such as adding excipients (helper lipids like small molecularphospholipids (e.g. DOPE and DSPC) and macromolecules lipids (e.g.PEG-DSPE and PEG-Ceramide)) into the LNPs and/or using more controllableself-assembly procedures, improved transfection efficiency of the fullysubstituted OCholB LNPs could be achieved.

Next, the MTT assay was conducted to examine the cytotoxicity ofmRNA-loaded nanoparticles against HeLa cells. As shown in FIG. 14E, itwas revealed that after 24 h of exposure (lipidoid/mRNA=10/1;[mRNA]=0.86 μg mL⁻¹), even though highest GFP⁺ cells percentage wasobtained from mRNA/Lpf2k treated group (FIG. 14D), the mRNA/Lpf2kcomplex showed significant cytotoxicity against HeLa cells, as 37.5%cell viability was recorded. On the other hand, all of the GFP mRNAloaded OCholB LNPs showed negligible cytotoxicities under the sameconditions (e.g. the cell viabilities were determined to be 84.7%, 94.5%and 100.1% for mRNA/75-OCholB, mRNA/76-OCholB, and mRNA/77-OCholBincubated samples), which is consistent with previous toxicity studiesof blank OCholB LNPs (FIG. 10E). This result indicate that the excellentcompatibility of OCholB LNPs and the possibility to further increase theintracellular delivery efficiencies by increasing the total dosageand/or exposure time of the mRNA/LNPs complexes. From the bight fieldimages io shown in FIG. 14F, it is obvious that after 24 h of exposure,significant morphological changes were observed from mRNA/Lpf2k treatedcells, while no obvious variations were observed for both the mRNA/LNPs(mRNA/75-OCholB, mRNA/76-OCholB, and mRNA/77-OCholB) and naked mRNAtreated cells, comparing the untreated control group. This result isconsistent with the cell viability study as shown in FIG. 14E andfurther validated the is advantage of OCholB LNPs as relative safetransfection nanocarriers.

Next, the possibility of using OCholB LNPs to deliver mRNA for genomeediting (Cre-loxP and CRISPR/Cas9 systems) purposes was examined. First,Cre mRNA was complexed with OCholB LNPs and tested against HeLa-DsRedcells. The HeLa-DsRed cells express red fluorescent protein, DsRed, onlyupon Cre protein-mediated recombination. After 24 h of incubation withmRNA/LNPs (lipidoid/mRNA=10/1; [mRNA]=0.86 μg mL⁻¹), the DsRed⁺ cellportions were determined by flow cytometry. As shown in FIG. 14G,75-OCholB, 76-OCholB, and 77-OCholB were all effective, as 67.2%, 48.7%and 72.8% of DsRed⁺ cells were recorded, respectively. Then the Cas9mRNA that expresses a version of Streptococcus pyogenes SF370 Cas9protein with an N and C terminal nuclear localization signal (NLS) wereloaded into LNPs, along with single-guide RNA (sgRNA) that targets asequence on GFP gene. GFP-HEK cells which steadily express GFP proteinswere used a cell model in this case. GFP -cells, indicating a successfulCas9-mediated knockdown of GFP expression, were analyzed using flowcytometry. After 48 h of incubation (lipidoid/mRNA/sgRNA=10/1/1; [mRNA]=[sgRNA]=0.86 μg mL⁻¹), it was found that all the three tested LNPs(75-OCholB, 76-OCholB and 77-OCholB) were unable to induce any evidentGFP knockout under this condition. The GFP⁻ cells portions recoded formRNA and sgRNA loaded 75-OCholB, 76-OCholB and 77-OCholB LNPs treatedGFP-HEK were 6.1%, 7.7% and 4.4%, respectively, which are comparable tothat of untreated control cells (7.0%). The intracellular deliveryresults of GFP, Cre and Cas9 mRNA molecules as shown in FIG. 14D and 14Gindicated that the efficacies of mRNA/LNPs are dependent on both of thetested cell types and the functions of protein expressed by cargo mRNAmolecules. This was also found to be applicable to protein delivery inour previous studies.

In order to further demonstrate the potentials of newly developed OCholBLNPs library in intracellular delivery applications, formulationoptimization was explored for improved Cas9 mRNA delivery for genomeediting. In this context, two strategies were tested, i.e., synthesizingnew OCho1B-tailed lipidoids with single tail rather than fullsubstitution, and adding helper lipids (phospholipids) into fullysubstituted OCholB LNPs. Single-tailed lipidoids, 75-OCho1B-1,76-OCho1B-1 and 76-OCho1B-1 were synthesized at first following similarprotocols as described before and characterized by ESI-MS([75-OCho1B-1+H]⁺, 736.55; [76-OCho1B-1+H]⁺, 734.64; [77-OCho1B-1+H]⁺,748.73). Nanoparticles were than fabricated using the samesonication/vortex procedures and the obtained LNPs were measured by DLS(75-OCho1B-1, <D_(h)>=302.6 nm, μ₂/

=0.30; 76-OCho1B-1, <D_(h)>=294.5 nm, μ₂/Γ²=0.30; 77-OCho1B-1,<D_(h)>=254.2 nm, μ₂/Γ²=0.33). The delivery efficacies of thesingle-tailed LNPs were first tested using GFP mRNA against HeLa cells(lipidoid/mRNA=10/1; [mRNA]=0.86 μg mL⁻¹; 24 h of exposure). 69.4%,72.5% and 68.9% of GFP⁺ cells were determined for mRNA/75-OCho1B-1,mRNA/76-and mRNA/77-OCho1B-1 treated HeLa cells, respectively. Cre mRNAcould also be efficiently delivered into HeLa-DsRed cells, as 87.3%(mRNA/75-OCho1B-1), 82.8% (mRNA/76-OCho1B-1) and 81.5%(mRNA/77-OCho1B-1) of cells were determined to be DsRed⁺ after 24 h ofexposure (FIG. 14G). However, it was found that the Cas9 mRNA and sgRNAcomplexed single-tailed OCholB LNPs also induced negligible GFP knockoutagainst GFP-HEK cells. Single-tailed OCholB lipidoids showed comparableor slightly higher delivery efficacies regarding to GFP and Cre mRNA,while failed with Cas9 mRNA and sgRNA delivery, similar to theirtwo-tailed counterparts. We then tried to add helper lipids into theoriginal two-tailed OCholB lipidoid nanoparticles formulations. As aproof-of-concept, DOPE was mixed with OCholB lipidoids(lipidoid/DOPE=1/1, weight ratio) and nanoparticles were fabricated(noted as 75-OCho1B-F, 76-OCho1B-F, and 77-OCho1B-F) and loaded withCas9 mRNA and sgRNA (mRNA/sgRNA=1/1, weight ratio). As shown in FIG.14H, after 48 h of exposure (OCholB lipidoid/mRNA=10/1; [mRNA]=0.86 μgmL⁻¹), 76-OCho1B-F showed similar GFP - portion (6.4%) as untreated(6.3%) and naked Cas9 mRNA and sgRNA treated (5.6%) cells. However,increased amount of GFP knockout cells was recorded for 75-OCho1B-F and77-OCho1B-F treated GFP-HEK cells, as 15.3% and 12.9% of the cells weredetermined to be GFP⁻. These results indicated that nanoparticleformulation optimization could be an effective strategy to achieveimproved performances. Overall, it was noted that the GFP knockoutefficacies of mRNA loaded OCholB LNPs were relatively low when comparedto our previously reported ribonucleoprotein (RNP) delivery results;however, by further molecular design (e.g. incorporating new types ofcationic amine head groups to expand the combinatorial library) andsupramolecular structural optimization (e.g. screening different speciesas well as compositions of excipients, optimization of cargo/carrierratios and incubation conditions like exposure duration and dosage),optimized intracellular delivery and subsequent genome editingperformances could be expected.

Example 12: Intracellular Delivery of Genome-Editing Protein

Protein- and peptide-based therapeutics have attracted tremendousattention during last three decades owing to their relatively highspecificity and low off-target effects. Formulations for treatment ofcancer, infection, inflammation and degenerative diseases have beendeveloped. Effective intracellular delivery methods for proteins andpeptides could is further expand their therapeutic modalities. As theintracellular delivery of protein using OCholB LNPs has beensuccessfully demonstrated in the previous internalization study using(−30)GFP-Cre protein as cargo and GFP as the fluorescent reporter, thefunctionality study was conducted using HeLa-DsRed cell line and DsRedprotein as the fluorescent reporter.

In this context, the internalization mechanism of the (−30)GFP-Cre/LNPscomplexes was studied at first, by introducing different endocytosisinhibitors, i.e., sucrose (clathrin-mediated endocytosis inhibitor),methyl-β-cyclodextrin (M-β-CD, cholesterol-depleting agent), dynasore(dynamin II inhibitor) and nystatin (caveolin-mediated endocytosisinhibitor), following our previously reported procedures. As shown inFIG. 15A, the internalization efficiencies ([lipidoid]=6.6 μg mL⁻¹,R-30)GFP-Crel=100 nM; exposure duration=6 h) of all three testedprotein/LNPs ((−30)GFP-Cre/75-OCholB, (−30)GFP-Cre/76-OCholB,(−30)GFP-Cre/77-OCholB) were significantly suppressed by sucrose anddynasore. M-β-CD and nystatin, on the other hand, did not induce obvioussuppression of the cellular uptake of these nanoparticles. Thisindicates that clathrin and dynamin play important roles in the cellularuptake of these (−30)GFP-Cre protein complexed OCholB LNPs. Comparing toother combinatorial library studies, it is clear that even loaded withsame cargoes and tested against same cell line, different lipidoids withdifferent chemical structures could be internalized through verydistinct pathways. Next, the genome-editing efficiencies of(−30)GFP-Cre/LNPs were determined by flow cytometry after 24 h ofincubation (with 8 h of (−30)GFP-Cre/LNPs complex exposure). Threedifferent concentrations of protein/lipidoid complexes (25 nM/1.7 μgmL⁻¹, 50 nM/3.4 μg mL⁻¹, and 100 nM/6.6 μg mL⁻¹) were tested for eachlipidoid nanoparticle. As shown in FIG. 15B, naked (−30)GFP-Cre proteininduced negligible genome editing efficacy regardless of the proteinconcentrations; while all the tested nanoparticles including Lpf2kshowed a dose-dependent DsRed+cell percentage pattern, i.e., higherprotein concentration correlate with higher genome editing and DsRedexpression level. Three lipids were found to be less efficient atdelivery, namely 87-OCholB, 90-OCholBand 304-OCholB (which are alsoshowed to be inefficient for NR and mRNA delivery). All other fullysubstituted OCholB LNPs (75-OCholB, 76-OCholB, 77-OCholB, 78-OCholB,80-OCholB, and 81-OCholB) showed comparable or even much higher DsRed⁺cells than the positive control, Lpf2k. For example, the DsRed⁺ cellswere recorded as 13.5%/55.4%/88.8%, and 27.7%/47.8%/94.7% for(−30)GFP-Cre loaded 75-OCholB, 76-OCholB and 77-OCholB, respectively, atthe protein concentration of 25, 50 and 100 nM. In particular, six ofthe OCholB LNPs (75-OCholB, 76-OCholB, 77-OCholB, 78-OCholB, 80-OCholB,and 81-OCholB) out-performed Lpf2k when tested at 100 nM of(−30)GFP-Cre, which further showed the advantage of newly developedLNPs. Furthermore, the typical flow cytometry profiles of(−30)GFP-Cre/LNPs ((−30)GFP-Cre/75-OCholB, (−30)GFP-Cre/76-OCholB,(−30)GFP-Cre/77-OCholB),(−30)GFP-Cre/Lpf2k and naked (−30)GFP-Cretreated HeLa-DsRed cells were shown in FIG. 15C, from which the enhancedDsRed fluorescent signal intensities were observed for thenanoparticles-based delivery systems, which are consistent with theresults shown in FIG. 15B. Then, the cytotoxicity profiles of(−30)GFP-Cre/LNPs, (−30)GFP-Cre/Lpf2k and naked (−30)GFP-Cre atdifferent concentrations against HeLa-DsRed cells (8 h of exposure;(−30)GFP-Cre/LNPs=25 nM/1.7 μg mL⁻¹, 50 nM/3.4 μg mL⁻¹, and 100 nM/6.6μg mL⁻¹) are measured using MTT assay after 24 h of incubation. As shownin FIG. 15D, in general, for all the samples tested, higher (−30)GFP-Creconcentrations induced lower cell viabilities. All nine of proteinloaded fully substituted OCholB LNPs showed relatively high cellviabilities. For (−30)GFP-Cre/75-OCholB treated cells, the viabilitieswere determined to be 95.5%, 91.8% and 83.1%, at the proteinconcentration of 25, 50 and 100 nM, respectively; the numbers for(−30)GFP-Cre/76-OCholB and (−30)GFP-Cre/77-OCholB are 97.8%/98.2%/97.2and 100.9%/96.6%/98.6%. The (−30)GFP-Cre loaded 78-0Ch1B, 80-OChlB and304-OChlB treated cells showed 79.1-81.4% of viabilities at 100 nM ofprotein, while all other samples were demonstrated to be non-toxicagainst HeLa-DsRed cells under the tested conditions. In sharp contrast,Lpf2k showed sever cytotoxicity under the same conditions, as 58.9%,56.7% and 51.2% of cell viabilities were determined with theconcentration of (−30)GFP-Cre at 25 nM, 50 nM and 100 nM, respectively.Meanwhile, the morphology changes of HeLa-DsRed cell treated withdifferent nanoparticle formulations were studied and the results areshown in FIG. 15E. It is clear that similar to the GFP mRNA loaded Lpf2ktreated HeLa cells (FIG. 14F), the HeLa-DsRed cells exposed to(−30)GFP-Cre/Lpf2k were unhealthy and shrunk; while those treated withOCholB LNPs ((−30)GFP-Cre/75-OCholB, (−30)GFP-Cre/76-OCholB and(−30)GFP-Cre/77-OCholB) were less affected comparing to the untreatedand naked protein treated control groups. The DsRed⁺ cell percentage wasthen plotted against the corresponding cell viability for all testedconditions (11 samples with 3 different concentrations), as shown inFIG. 15F, with dotted lines denoting 80% of cell viability and 50% ofgenome editing io efficacy (DsRed⁺ cell portion), respectively. Samplesfound in upper left quadrant are non-toxic and inefficient for delivery;samples in lower left quadrant are toxic and inefficient; samples inlower right quadrant are efficient but toxic; samples in upper rightquadrant are non-toxic and efficient, which would be top candidates forfurther study. It is clear that Lpf2k (shown in dotted purple circle) athigh concentration is relatively efficient for genome editing, whilealso toxic to the target cells. On the other hand, most of the(−30)GFP-Cre loaded OCholB LNPs are almost non-toxic when compared to(−30)GFP-Cre/Lpf2k, similar to naked (−30)GFP-Cre protein. 87-OCholB,90-OCholB and 304-OCholB (shown in dotted dark blue circle) are lessefficient for genome-editing; while high genome editing efficacy andexcellent tolerability were achieved by using 75-OCholB, 76-OCholB and77-OCholB (shown in dotted green circle). Above all, these resultsindicated that the newly developed OCholB LNPs could serve as highlyefficient and safe nanocarriers for Cre recombinase protein delivery forin vitro genome editing.

Example 13: In Vivo Toxicity Study

Both the blank (FIG. 10E and 13C) and cargo (GFP mRNA and genome editingprotein) loaded (FIG. 14E and 15D) OCholB LNPs showed relative highbiocompatibility in vitro. The in vivo toxicity of the OCholB LNPs wasfurther examined by measuring body weight change and biologicalfunctions of kidney and liver through serum biochemical tests usingBalb/c mice. 4-6 weeks old Balb/c mice (n=3) were injected with blank75-OCholB, 76-OCholB and 77-OCholB LNPs (50 μg LNPs for each injection)through tail vein at day 1 and day 5, body weights were monitored for 14days, and blood were collected and analyzed at day 14. As shown in FIG.16A, comparing to the untreated control group, the body weights of LNPs(75-OCholB, 76-OCholB and 77-OCholB) injected mice showed negligibledifferences throughout the study. Serum concentrations of creatinine,urea, aspartate aminotransferase (AST), and alanine aminotransferase(ALT) of LNPs injected mice were very similar to control mice (FIG.16B).

These results indicated that these OCholB LNPs would not inducesignificant body weight change or serve organ damages through systemicadministration under the tested conditions, indicating these LNPs couldbe used as safe carriers for in vivo delivery purposes.

EXAMPLE 14: In Vivo Protein and mRNA Delivery for Genome Editing

Cre-loxP system and transgenic Ai14 mouse model were used in the in vivogenome editing study. As shown in FIG. 17A, this mouse model has agenetically integrated loxP-flanked STOP cassette that prevents thetranscription of red fluorescent protein, tdTomato. When the Crerecombinase mediated gene reorganization occurs, the STOP cassette couldbe removed, resulting in the expression of fluorescent tdTomato reporterprotein.

Local delivery through intramuscular injection (IM injection; rear leg)using (−30)GFP-Cre protein and Cre mRNA loaded 76-OCholB LNPs (FIG.17B). Ai14 mice (n=3) received single dose of (−30)GFP-Cre/LNPs (50 μgof protein) or mRMA/LNPs (10 μg of mRNA) injection at day 1 and weresacrificed at day 10. Skeletal muscles were collected, fixed,cryosectioned and imaged for tdTomato expression analysis (seeexperimental sections). As shown in FIG. 17D and 17E (Blue channel,DAPI; Red channel, tdTomato), contrary to untreated control muscle,strong tdTomato fluorescent signals from both (−30)GFP-Cre /LNPs andmRNA/LNPs injected muscles were recorded. A larger portion of tdTomatopositive cells were found in the protein/LNPs injected muscle samplesthan the mRNA/LNPs counterpart.

The OCholB LNPs was further investigated if they can induce successfulgene editing in vivo through a systemic administration pathway. Atfirst, Ai14 mice (n=3) were injected through tail vein (intravenous (IV)injection) with (−30)GFP-Cre protein loaded LNPs at day 1 and 5 (50 μgprotein for each injection; 100 μg in total), then sacrificed at day 14for analysis (FIG. 17C). In this case, five of the top OCholB LNPs thathave been demonstrated to be effective in vitro as shown in FIG. 17F aretested, i.e., 75-OCholB, 76-OCholB, 77-OCholB, 78-OCholB, 80-OCholB. Theheart, liver, spleen, lung and kidney from each group were collected andanalyzed. Relative high genome editing efficacy was achieved in the lungand spleen of (−30)GFP-Cre/80-OCholB and (−30)GFP-Cre/76-OCholB injectedAi14 mice, respectively, as shown in FIG. 17F. Like most intravenousnano-therapeutics, the (−30)GFP-Cre protein complexed OCholBnanoparticles injected through tail vein would travel first to theheart, and from there directly to the lung. The nanoparticleformulations (which may have complexed with serum proteins) could beeasily trapped in the vasculature structures in the capillary bed of thelung, delaying or inhibiting the redistribution of LNPs to the liver,spleen and other organs. During the in vivo transport and redistributionprocess, some of the protein loaded LNPs may successfully enter into thecells in lung or spleen to induce the genome editing and tdTomatoexpression cascade. Degradation, aggregation and/or immune cellssequestration of the cargo protein and carrier LNPs would dramaticallyreduce or perhaps even prohibit genome editing events. Nevertheless,80-OCholB and 76-OCholB were demonstrated to be efficient for(−30)GFP-Cre protein delivery into lung and spleen, respectively, invivo through systemic administration.

Next, in vivo systemic mRNA delivery using OCholB LNPs was tested usinga similar intravenous injection protocol (FIG. 17C). Cre mRNA loaded76-OCholB LNPs were injected at day 1 and day 5 (10 μg mRNA for eachinjection; 20 μg in total), and mice were sacrificed at day 14. All themajor organs (heart, liver, spleen, lung and kidney) were collected andanalyzed. As shown in FIG. 17G, significant amount of tdTomato positivecells were recorded in the spleen, and positive signals were not foundin other organs. It was noted that both of the (−30)GFP-Cre protein andCre mRNA loaded 76-OCholB LNPs induced genome editing in the spleen,which indicated that the nature of carrier lipidoids may impact themetabolism and biodistribution of the whole delivery system. Overall,the genome editing efficacies of systemically administratednanoparticles (both of the (−30)GFP-Cre protein and Cre mRNA loadedLNPs) seemed much lower than that of local injection, which isunderstandable as the formulations injected through vein would encountermuch more physical as well as biochemical barriers. However, it is stillworth pursuing as systemic administration supplies a wide range ofpossibilities for the treatment of human conditions or diseases. Aboveall, these in vivo genome editing results suggested the possibility ofusing OCholB LNPs as nanocarriers to deliver functional proteins as wellmRNA in vivo both through the systemic and local administrations routesfor genome editing purposes.

Materials and Methods General

The chemicals used for lipidoids synthesis, amphotericin B andcommercial kits used to assess hepatotoxicity and nephrotoxicity werepurchased from Sigma-Aldrich.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000](DSPE-PEG2000 Amine) was ordered from Avanti. HEK293 cellswere cultured in Dulbecco's modified eagle's medium (DMEM,Sigma-Aldrich) with 1% penicillin-streptomycin (Gibco) and 10% fetalbovine serum (FBS, Sigma-Aldrich). Hydrodynamic sizes and polydispersityindexes (PDI) of AmB nanoparticles were measured by Zeta-PALS particlesize analyzer (Brookhaven Instruments). The concentration, RBC hemolysisand cell viability of AmB encapsulates were measured by SpectraMax M2emicroplate readers. The AmB encapsulates were lyophilized by freezeDryer (Labconco). Human whole blood was ordered from Reaserch BloodComponent, LLC. The strain of C. albicans (SC₅₃₁₄) was obtained from thelaboratory of Professor Carol A. Kumamoto in Department of MolecularBiology and Microbiology of Tufts medical center. Tissue samples (100mg) were ground by bead bug microtube homogenizer (Benchmarkscientific). The plasma and tissue concentration of AmB were measured byhigh performance liquid chromatography (HPLC) (Agilent 1200) inchemistry department of Tufts University. Female BALB/c mice (age 6-8weeks, weight 20-30 g) and female Sprague Dawley rats (age 8-10weeks,weight 200-250 g) were ordered from Charles River. The animal protocolof this study was approved by the Institutional Animal Care and UseCommittee (IACUC) of Tufts University (B2018-73) and all in vivoexperiments were performed under the approved animal care guidelines.

Preparation of AmB nanoparticles.

The AmB encapsulates were prepared as follows: Briefly, 1 mg eachlipidoid was mixed with 1 mg AmB which was already dissolved in 300 μlDimethyl sulfoxide (DMSO). The mixtures were sonicated for 30 minutesand then vortexed for 10 minutes until each was completely dissolved.Then the AmB nanoparticles were formulated with 10 mg/mL DSPE-PEGdissolved in ethanol with the mole ratio of 1:6.8 (DSPE-PEG tolipidoids). As a control group, the AmB nanoparticles were notformulated with DSPE-PEG. Each solution was added drop-wise to a glassbottle containing 600 μl sodium acetate buffer (pH 5.0) with continuoushomogenization at 700 rpm. Then the solutions were further dialyzedagainst distilled water by using the dialysis bag (MWCO: 3500Da) for 4 hto remove the DMSO and sodium acetate buffer with a stirring speed of600 rpm/min The AmB encapsulates were transferred to 2m1 glasses bottlesto observe their visual transparency for 2 weeks. The data recorded arethe mean of three experiments carried out independently.

Stability and Particle Size

The particle sizes and PDI were assayed by dynamic light scattering(DLS), 1 and 2-week endpoints to evaluate the stability of AmBnanoparticles. Mean size (nm) and PDI were determined based on sizedistribution by number. The AmB nanoparticles were dispersed indeionized water with 10-fold dilution before measurement. Three runs of60 s per sample were carried out at a detection angle of 90° in the sameconditions. All nanoparticles were prepared and measured in triplicate.

Drug Loading Content

In order to quantify the amount of AmB loaded, regression calibrationcurve of AmB concentration was calculated by studying the absorbance ofdifferent AmB concentrations (0.001-1.0 mg/mL) dissolved in DMSO bySpectraMax M2e. The wavelength ranging from 300 to 450 nm was selectedfor UV-Vis absorbance spectrum. The amounts of AmB encapsulated intoliposome were determined by dissolving the nanoparticles in DMSO andthen their absorbance at 392nm wavelength were measured. The drugloading content (DLC) of AmB was calculated according to linearregression calibration curve and then the following equation: Drugloading content (%)=W_(loaded)×100/W_(polymer)+W_(loaded), whereW_(loaded) is the weight of AmB loaded in the liposomes afterencapsulation, and W_(polymer) is the weight of lipidoids. The datarecorded are the mean of three experiments carried out independently.

In Vitro Antifungal Activity

The minimum inhibitory concentration (MIC) and C. albicans (SC₅₃₁₄)strain were used to test antifungal efficacy of AmB encapsulates invitro according to the Reference Method for Broth Dilution AntifungalSusceptibility Testing of Yeasts. Briefly, the yeast was grown onSabouraud Dextrose Agar (SDA) plates and inoculated into water to yielda final inoculum concentration of 1-5×10⁶ yeast cells/mL. C. albicanscells suspension was diluted 1:20 in RPMI-MOPS growth medium and 100 μldispensed into a microliter tray containing a serial concentration ofAmB range from 0.125 to 32 μg/mL and 0.109375 μg/mL to 14.0 μg/mL. Threewells containing drug-free medium and inoculum were used as positive andnegative controls. The inoculated plates were incubated at 35° C. for 48h. The growth in each well was visually estimated at 24 h and 48 h. TheMIC was recorded to be the lowest concentration of AmB that preventedvisible growth of C. albicans and expressed in μg/mL. The data recordedare the mean of three experiments carried out independently.

Hemolysis Test from Human Erytrocytes

In order to screen the optimized AmB encapsulates, high doses of AmBencapsulates were needed in toxicity evaluation. The AmB encapsulateswere lyophilized by cryoprotectant, then were reconstituted to properlyvolume with filtered deionized water followed by shaking to gethomogenous liposomal dispersion. The hemolysis was performed aspreviously described. Venous blood obtained from a healthy volunteerstored at 6±2° C. Whole blood was centrifuged (30 min at 1,600×g) andthe supernatant was pipetted off and discarded. RBCs were then washedthree times with isotonic PBS of pH 7.4 and were finely dispersed in PBSat 2% stock solution. Subsequently, 90 μl of the RBCs suspension weremixed with 10 μl of PBS containing different AmB encapsulates, free AmBand Fungizone® in triplicate. The final AmB concentration was 200, 100,50 and 25 μg/mL respectively in all nanoparticles. Each sample was thenincubated at 37° C. After 1 h incubation, hemolysis was stopped and RBCsnot lysed were removed by centrifugation (5 mM at 5000×g). Thesupernatants were collected for analysis to determine the extent ofhemolysis by reading the absorption of hemoglobin at 540 nm bySpectraMax M2e. Hemolysis (%)=(Ab_(s)−Abs₀)×100/(Abs₁₀₀−Abs₀), where Absis the absorbance of AmB encapsulates, Abs₁₀₀ is absorbance of the 100%lysed sample treated with 1% Triton X100 sample and Abs₀ is theabsorbance of unlysed sample treated with PBS.

In Vitro toxicity in Mammalian Cells

Human embryonic kidney HEK293 cells were used to evaluate cell viabilityof AmB encapsulates. The cells were transferred to 96-well tissueculture plates at 5×10³ cells per well and incubated for 24 h at 37° C.prior to drug treatment which containing different concentrations of AmBencapsulates, free AmB and Fungizone® (equivalent of AmB 200, 100, and25 μg/mL). 30 μl of MTT stock solution (5 mg/mL) was added to each welland the plates were incubated for 4 h at 37° C. After discarding theculture medium, 200 μl DMSO was added to dissolve the blue formazancrystals converted from MTT. Cell viability was assessed by measuringthe absorbance at 570 nm by SpectraMax M2e. The cell viability wasexpressed as percentage calculated with the absorbance obtained fromcontrol well without drug treatment using the following equation: Cellviability (%)=Abs_(t)/Abs_(c)×100%, where Abst is the absorbance ofdrug-treated well and Abs_(c) is the absorbance of control well withoutdrug treatment.

Pharmacokinetics Analysis Studies

For this experiment, six female Sprague Dawley rats were fastedovernight for about 12 h with free water access and were dividedrandomly in two groups. Considering the maximum tolerated dose(MTD) ofFungizone® is 2 mg AmB/kg, the rats were intravenously administered viatail vein with either screened AmB encapsulate or Fungizone® at a singledose equivalent of AmB 2 mg/kg. The blood samples (˜0.5m1) of each groupwere collected in heparinized tubes by retro-orbital puncture at eachtime point (10, 30 mM and 1, 2, 4, 6, 8, 12, 24, 36 h) afteradministration. Each blood sample was centrifuged at 10000 rpm for 10 mMand plasma was collected for the determination of the AmB concentration.Two parts of methanol was added into one part of the plasma. Themixtures were vortexed for 5 mM followed by centrifugation (13000 g, 4°C. and 30 mM). The supernatants were collected for HPLC as describedpreviously. HPLC analysis of each sample was performed with a modularliquid chromatograph system (Agilent™). The mobile phase consisted ofacetonitrile and 10 mM sodium acetate buffer, pH 4.0 (40:60, v/v) andthe flow rate kept at 1 mL/min. Compounds were separated on a 4.6×100mm, 3.5 μm size eclipse plus C₁₈ reverse-phase column. The relativeretention time of AmB was 4 min. The effluent was monitored at 408 nm.Plasma AmB concentrations were calculated from linear regressioncalibration curves. Non-compartment pharmacokinetic analysis of Pkssoftware designed by Zhang was used to evaluate the AmB plasmaconcentrations versus time data.

Tissue Biodistribution Test

Twenty-four BALB/c mice were randomly divided into four groups (n=6) forthe tissue distribution study. Three groups were injected with screenedAmB encapsulates via tail vein at a single dose of 10 mg, 5 mg, 2 mgAmB/kg respectively. One group was intravenously injected io withFungizone® at a single dose of 2 mg AmB/kg. Three mice of each groupwere sacrificed by CO₂ inhalation, and tissues (liver, spleen, lungs,kidney, heart and brain) were taken out at 48 h and 72 h postadministration respectively and kept at −80° C. until they were furtherprocessed. Tissue samples (100 mg) were ground and homogenized with 200μl DI water in a high-speed by bead bug tissue homogenizer (2 min, 4000rpm). Two parts of methanol were added into one part of the homogenate.The resulting mixtures were vortexed for 2 min followed bycentrifugation (13000 g, 4° C. and 30 min). The supernatants were usedfor HPLC analysis in the same way as pharmacokinetics analysis.

Hepatotoxicity and Nephrotoxicity Tests

Fifteen female BALB/c mice were randomly divided into five groups (n=3).Three groups were injected with screened AmB encapsulate via tail veinat a single dose of 10 mg, 2 mg AmB/kg, respectively. One group wasadministrated in the same way with Fungizone® at single a dose of 2 mgAmB/kg. The control group was injected with PBS. The blood samples (-0.2mL) were collected by the mandibular vein puncture at 48 h and 72 hafter injection and were allowed to coagulate at 4° C. and thencentrifuged for 10 min at 5000 rpm to collect the serum. Kidney andliver biochemical parameters were performed as per the manufacturer'sguidelines to analyse the nephrotoxicity and hepatotoxicityinvestigations including Creatinine (Cr), Blood urea nitrogen (BUN),Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST). Theconcentrations were calculate based on the regression calibration curvesof each kit.

Statistical Analysis

All data expressed as mean ±standard deviation (SD). The differenceamong the groups was evaluated by two-way analysis of variance (ANOVA)followed by the Turkey-Kramer multiple comparison test for more than twogroups, and student t-test for comparing two groups using Prism software(Graph Prism7.0 Software Inc. CA, USA). The differences were consideredsignificant when p<0.05. Whereas*p<0.05 and ** p<0.001 versus controlgroup described in the legends.

Example 15: Optimization of AmB Lipidoids Encapsulates and StabilityEvaluation

AmB is poorly soluble in aqueous and organic solvents. Its watersolubility at physiological pH is less than 1 mg/L. The amphipathicproperty rendered a challenge for efficient and economical deliver. Theamphipathic characteristic rises from the apolar and polar sides of thelactone ring, while the amphoteric property is due to the presence ofionizable carboxyl and amine groups (FIG. 18 a ). AmB was firstformulated with different to lipidoids 75-O14B, 78-O14B or 87-O14B,opaque suspensions were obtained, but all precipitated in less than 1week as show in FIG. 2 . The particle sizes increased dramatically andthe PDI increase to more than 0.7 at the end of 2 weeks. As we all know,the particle size plays an important role in pharmacokinetics andtoxicity. Because particle size larger than 100 nm in diameter is easilyinteracts with plasma proteins, then can be easily recognized by RES andeliminated more rapidly from blood such as Amphocil®. However, too smallparticle size could increase glomerular filtration and drug renalexcretion such as Fungizone®. The optimizable size of nanoparticle is50-100nm.

To increase the solubility and stability, AmB was either formulated withDSPE-PEG or encapsulated in QLDs. As a result, the nanoparticlesdemonstrated better drug solubility with more clearly yellowish colortranslucent solutions, but still a little cloudy when formulated withDSPE-PEG2000 (FIG. 19 ). The particle sizes increased to 500-900 nm andPDI increased to more than 0.5 at the end of 2 weeks (FIG. 20 a and 20 b). However, when AmB was loaded by QLDs, homogenous transparent yellowsolutions were obtained and remained stable in the following 2 weeks(FIG. 2 ). The particle sizes were decreased to100-160 nm, but still alittle higher than the optimizable size of nanoparticles (FIG. 20 a and20 b ). further formulated DSPE-PEG with QLDs to encapsulate AmB. Theparticle sizes of AmB/Q75-O14B-F and AmB/Q78-O14B-F decreased to 70-100nm (FIG. 20 a ). Although the particle size of AmB/Q87-O14B-F was alittle high(110-120 nm), but it still decreased as compared toAmB/Q87-O14B. The quaternized liposomal vesicles either formulated withDSPE-PEG or not were all homogeneous and similar in nature with regardsto particle size and PDI after preparation even following 2 weeks period(FIG. 20 b ).

QLDs and DSPE-PEG enable the formation of stable AmB encapsulates andfacilitate produce smaller condensed structure, in which AmB wasintercalated between the lipid bilayer (FIG. 18 a ). The stability ofliposome depended on the nature of the phospholipid molecules containedin their structure. QLDs having two quaternized amine headscharacterized by its higher solubility, easier and economiccombinatorial synthesis as well as higher delivery efficiencies, whichmake the QLDs attractive. PEG is biocompatible, but a large amount isneeded for the water-soluble AmB complex. QLDs increased the AmBsolubility property and decreased the amount of DSPE-PEG with 1:6.8PEG-to-lipidoid molar ratio. The lipidoids were quaternized by beingdissolved in THF and reacting with excessive amount of methyl iodideovernight at room temperature in the dark. The precipitations werefiltered, washed with diethyl ether then dried in vacuum (FIG. 18 a ).Another superior aspect of AmB encapsulates described here is less-costexcipients and easier preparation as compared to Ambisome®. Ambisome® isformulated with injectable good manufacturing practice (GMP)-gradecholesterol because of the agents of bovine spongiformencephalopathy/transmissible spongiform encephalopathy and the relatedanalysis procedure, which make the final product expensive.

Example 16: Drug Loading Content

AmB dissolved in DMSO exhibited three main spectrophotometric peaks inthe UV range consistent as previously reported. AmB concentrations weremeasured by absorbance of properly diluted ratio at 392 nm andcalculated by calibration curve with correlation coefficient equivalentto 0.9985. The DLC of AmB encapsulates were 38.9-49.9% indicatingexcellent association of AmB with the liposomes as show in FIG. 21 a .AmB/Q78-O14B-F showed the highest DLC about 49.9% among theseencapsulates. The DLC efficiency depending on the polarity and partitioncoefficient determined its localization in liposomal membrane. Becausethe AmB is amphipathic, it resides in the acyl hydrocarbon chain,adjacent to the water-lipid interface (FIG. 18 a ).

Example 17: In Vitro Antifungal Activity

AmB has high affinity to ergosterol in fungal cell membrane, leading tothe pore formation, intercellular ion leakage and untimately fungal celldeath. The MIC test with 24 and 48 h incubation showed the lower MIC forthe all AmB encapsulates when compared to 30 free AmB and Fungizone®against yeast strains C. albicans (SC₅₃₁₄) (FIG. 21 b ). The MIC ofFungizone® was 0.875 μg/mL and free AmB was 1.75 μg/mL after 48 hincubation against C. albicans (FIG. 21 b ), consistent with the resultsobtained by Radwan. Among the all AmB encapsulates, AmB/Q78-O14B-Fpresent the lowest MIC (0.29±0.13 μg/mL), which was almost 6-fold lowerthan free AmB, 3-fold lower than Fungizone® as shown in FIG. 21 b (p<

The structure characteristic of the quaternary amino group maycontribute to the higher antifungal efficacy by increasing AmBconcentrations in fungal cell membranes and the synergistic antifungaleffect with AmB.

Example 18: Hemolysis Test from Human Erytrocytes (RBCs)

To evaluate the toxicity of AmB encapsulates, hemolysis induced bydifferent concentrations of AmB were compared with free AmB andFungizone®. Free AmB exhibited almost 80.69±2.39% and 102.47±1.04% ofhemolysis at 100 and 200 μg AmB/mL, respectively (FIG. 22 a ).Fungizone® showed almost 52.05±9.83% and 68.84±10.28% of hemolysis atthe same concentration of AmB. The hemolytic properties of AmBencapsulates were little affected up to 200 μg AmB/mL exceptAmB/Q75-O14B-F encapsulates which exhibit 21.39±3.58% at 200 μg AmB/mLas shown in FIG. 22 a (p<0.05). Therefore, AmB encapsulates were lesshematotoxic than Fungizone®, because AmB released from bilayerunilamellar was lower than from micellar formulation. The micelles ofFungizone® are a relatively weak barrier compared to lipid bilayers andthe drug in Fungizone® is more available than AmB encapsulates,resulting in faster leakage of hemoglobin and potassium. Another reasonfor increased of hemolysis for Fungizone® is that the component ofsodium deoxycholate which acts as a surfactant can induce hemolysisitself.

Example 19: In Vitro Toxicity in Mammalian Cells

FIG. 22 b showed the cell viabilities of AmB encapsulates, Fungizone®and free AmB at concentrations ranging from 25 to 200 μg AmB/mL. FreeAmB and Fungizone® showed obvious cytotoxicity to HEK293 only after 24 hincubation even at low concentration. After formulated with the QLDs,the cell viabilities of AmB/(Q75-O14B, Q78-O14B, Q87-O14B) encapsulateswere slight decreased compared to AmB/(75-O14B, 78-O14B 87-O14B)-Fencapsulates. Simultaneously, the cell viability of all AmBnanoparticles dramatically increased when compared to Fungizone® andfree AmB (p<0.05). After formulated with DSPE-PEG, the cell viabilitiesof AmB/(Q75-O14B, Q78-O14B, Q87-O14B)-F encapsulates remained at 70-80%up to 200 μg AmB/mL. This perhaps contributed to biocompatible andrelatively nontoxic DSPE-PEG which is capable of interacting with thepositive amino group of AmB to form an ionic complex in the bilayers.Another reason is that QLDs effectively encapsulated AmB resulting inslow and sustained AmB release and reducing the toxicity.

Based on the results from the in vitro evaluation, the AmB/Q78-O14B-Fdemonstrating minimally toxicity, MIC and most stability was finallyscreened to be the most effective delivery system for further analysisin vivo.

Example 20: Pharmacokinetics Analysis Studies

Pharmacokinetics impact the accumulation of the drug in the tissues.AmB/Q78-O14B-F and Fungizone® were intravenously injected into rats at adose of 2 mg AmB/kg body weight for comparison of their pharmacokineticprofiles. The estimated plasma concentration-versus-time profiles wereshown in FIG. 23 a and corresponding mean pharmacokinetic parameters iowere summarized in Table 1.

The results demonstrated that plasma concentration profiles of bothAmB/Q78-O14B-F and Fungizone® showed a rapid initial distributive phase.Meanwhile, AmB/Q78-O14B-F yielded higher maximal plasma concentration(Cmax) for AmB than Fungizone® (25.13±7.05 and 2.66±0.81 μg/mL,respectively, p<0.05)(Table 1). The AmB concentration of Fungizone®could not detectable in all rats at 24 h and in one rat at 12 h. The AmBwas still detectable at 24 h (0.74±0.12 μg/mL) after administration andremain above the MIC (0.39±0.13 μg/mL). AmB would show fungistaticactivity if the concentration is less than 0.5 to 1-fold MIC and performstrong fungicidal activity when its concentration is more than 0.5 to 1time of the MIC. The results indicated that AmB/Q78-O14B-F still havefungicidal activity after 24 h administration beneficial for blood-borneinfection such as disseminated candidiasis.

Moreover, AmB/Q78-O14B-F showed higher AUC (46.58 ±6.28 mg*h/L) over4-fold against that of Fungizone® (10.98±5.02 mg*h/L) and the smallervolume of distribution (Vd) (177.08±46.05L/kg) almost half against thatof Fungizone® (296.86±12.02 L/kg) (p<0.05) (Table 1). Thepharmacokinetic behavior of AmB/Q78-O14B-F seems to be similar toAmbisome® which also exhibits a high Cmax, AUC, slow CI and small Vd.One explanation is that amino group of AmB, with its positive chargeforms an ionic complex with QLDs. This mechanism thereby promotes theretention of AmB within the liposomal bilayer and released it slowly,resulting in a longer circulation in blood. Another reason is DSPE-PEGpossesses properties of its biocompatibility and varied conformationalflexibility which prolongs blood circulation time by being attaching onthe surface of anionic lipids and thus further facilitates the retentionof AmB within bilayer.

It is very important to avoid the uptake by RES and prolong the plasmacirculation time to improve the distribution and effect when theinfected target is a tissue except for liver and spleen. Fungizone®displayed low AUC, Cmax, large CI and wide Vd (Table 1) consistent withpreviously reported results. The low AmB plasma concentration ofFungizone® could be explained by the fast release of AmB from micellarformulation of Fungizone® and high uptake of AmB by RES of the liver andspleen. We also observed an interesting phenomenon that Fungizone®displayed a second peak in plasma levels 4 h after administration whichhave already been reported respectively before by Swenson and Serrano(FIG. 6 a ). This may be related to the redistribution from the tissuessuch as liver.

TABLE 1 Pharmacokinetic parameters of AmB after intravenous injection ofAmB/Q78-O14B-F and Fungizone ® in rats at a dose of 2 mg AmB/kg.Parameters Fungizone ® AmB/Q78-O14B-F Dose (mg/kg) 2 2 AUC₀₋₂₄ (mg *h/L)  10.98 ± 5.02  46.58 ± 6.28* MRT (h)  27.92 ± 32.0  21.87 ± 5.48C_(max) (mg/L)  2.66 ± 0.81  25.13 ± 7.05** T_(1/2) (h)  19.11 ± 22.94 21.14 ± 6.91 CL (L/Kg/h)  19.04 ± 12.02  5.93 ± 0.98* Vd (L/Kg) 296.86± 159.06 177.08 ± 46.5* Note. All data represent as mean ± SD(n = 3).Abbreviations: AUC Area under the concentration time curve; MRT MeanResidence Time, Cmax Maximal plasma concentration, T_(1/2) half-life, CLclearance, V volume of distribution, *p <0.05 and **p <0.001 vsFungizone ®.

Example 21: Tissue Biodistribution Test

Once the nanoparticles leave blood circulation, it is very important toknow where the drug goes and how long it remains in a particular tissue,because tissues are also the primary site of systemic fungal infection.The results of tissues distribution after 48 h and 72 h intravenousadministration were shown in FIG. 23 b-23 e . All the mice were alivewhen administrated with AmB/Q78-O14B-F at a single dose of equivalent ofAmB 5 mg/kg and 2 mg/kg. Each group has one deceased mouse whenadministrated with the dose equivalent to AmB 10 mg/kg of AmB/Q78-O14B-Fand 2 mg/kg of Fungizone®

The results indicated that AmB/Q78-O14B-F exhibited lower concentrationsin liver (2.07±0.30 μg/g) and spleen(5.10±0.97 μg/g) compared to that ofFungizone® (5.80±1.43 μg/mL in liver and 6.25±1.30 μg/mL in spleen)after 48 h injection at a single dose of 2 mg AmB/kg(Figs. 23b and 23c).Because AmB/Q78-O14B-F avoided been immediate recognized by RES leadingto prolonged circulation in plasma. The recognition of particle ismediate by opsonization in blood, depending on the distance between theparticle and opsonins. When the distance is short like Fungizone®,opsonins bind to the surface of the particle then are recognizable byRES. Furthermore, the AmB concentration of AmB/Q78-O14B-F decline to theequivalent level in liver (1.46±0.06 μg/g) and spleen (1.37±0.06 μg/g)in comparison to 1( )Fungizone® (1.80±0.10 and1.23±0.14 μg/mL,respectively) after 72 h injection, and still remain above the MIC(FIGS. 23 b and 23 c ). The long-term tissue retention suggests that thedrug could be given intermittently, instead of daily, without losingefficacy and this would reduce the cost and possible toxic side-effects.Unfortunately, AmB/Q78-O14B-F exhibited none AmB distribution in braintissue, which was not beneficial for the intracranial fungal infectionsuch as cryptococcal meningitis.

We noticed that the AmB concentrations were low in kidneys (48 h,0.79±0.70 μg/g, 72 h, 0.45±0.39 μg/g) as compared with Fungizone®(48 h,1.93±0.23 μg/g; 72 h, 23e), indicating reduced distributions of AmB tokidneys. The explanation is that liposome is large enough to avoidglomerular filtration and drug renal excretion and led to reducednephrotoxicity of AmB encapsulates.

There was another superior attribute of this nanoparticle thatAmB/Q78-O14B-F accumulates in the lungs at higher concentrations thanFungizone® (2.96±1.06 vs 1.45±0.24 μg/g, respectively, p<0.05) (FIG. 23d ). After 72 h injection, the concentration in lung of AmB encapsulateswas 2.12±0.27 μg/g, however, low AmB concentrations were detectable inthe lungs of Fungizone® treated mice (p<0.05) (FIG. 23 d ). It isbeneficial for pulmonary fungal infection when the target ofAmB/Q78-O14B-F is lung site such as invasive aspergillosis.Unfortunately, AmB/Q78-O14B-F did not exhibit any distribution in braintissues, which was not beneficial for the intracranial fungal infectionsuch as cryptococcal meningitis.

An increase in the dose-dependent response was noted in the tissues ofAmB/Q78-O14B-F treated mice (FIG. 23 b -23e). When the injection dosewas increased to 5 mg AmB/Kg, higher concentrations were detected in allorgan tissues of mice. All mice were survived in the experiment, notoxicities were identified in subsequent in vivo toxicity test. However,one mouse died in 12 h when the dose of AmB/Q78-O14B-F was increased to10 AmB/kg. It means the toxicity increased when higher concentration ofAmB accumulated in the tissues post administration. Low concentrationsof AmB were found in heart tissues after 48 h administration at a doseof 10 mg AmB/kg. One mouse succumbed to Fungizone® at the single dose of2 mg Amb/kg intravenous administration. These results indicatedAmB/Q78-O14B-F have wider and safer therapeutic window as compared with

Fungizone®, which was confirmed in following in vivo toxicity test.

Example 22: In Vivo Toxicity Tests

In vivo toxicity evaluations, the results suggested AmB/Q78-O14B-F didnot affect io liver (ALT and AST) and kidney (Cr and BUN) functions atthe dose of either 2 mg or 5 mg AmB/kg treated mice compared to that ofthe control group (FIG. 24 ). The results were consistent with thereduction of AmB concentration accumulation in kidneys of 2 mg or 5 mgAmB/kg of AmB/Q78-O14B-F treated mice (FIG. 23 e ). Thus, glomerularfiltration is reduced and nephrotoxicity is minimized However,AmB/Q78-O14B-F increased the Cr and BUN is level or the liver enzymesAST and ALT when the dose was elevated to 10 mg AmB/kg and all havesignificant differences compared with that of the control group (p<0.05)(FIG. 24 ).

The hepatotoxicity and nephrotoxicity may relate to the AmB retention inkidney and liver after increasing dose administration. In comparison,Fungizone® induced significant increases in Cr, BUN, ALT and AST after72 h administration at similar dose of 2 mg AmB/kg when compared toAmB/Q78-O14B-F (p<0.05) (FIG. 24 ). The findings demonstrated thatAmB/Q78-O14B-F present a substantial reduction in toxicity and anincrease in the therapeutic window of AmB in comparison to Fungizone®.

Example 23: Lipids with Fluorine Chains

Synthesis

A fluorine-containing tail (2.5 equiv.) was mixed with an amine head (1equiv.) in a clean glass vial. The mixture was kept under 70 C withcontinuous stirring for 48 h. The reaction was then stopped, and thecrude product was purified via silica gel column chromatography, usingmethanol and dichloromethane as the mobile phase.

Assay

The results of the percentage of GFP positive and DsRed positive cellsfor the above different lipids with fluorine chain are summarized in abar graph in FIG. 25 and FIG. 26 .

Example 24: New Library 1—Amine 200 with Different Hydrophobic Tails

The results of the percentage of GFP⁺ cells for the above lipids withdifferent hydrophobic tails (synthesized from amine 200) are summarizedin a bar graph in FIG. 27 .

Example 25: New library 2—Cyclic Amine Analogues

The results of the percentage of GFP⁺ cells for lipids synthesized fromdifferent cyclic amine analogues are summerized in a bar graph in FIG.28 .

Example 26: New library 3—Imidazole Containting Amine Analogues

The results of the efficiency of mRNA delivery to CD8+ T cells for theabove lipids synthesized from different imidazole-containing amineanalogues are summerzied in a bar graph in FIG. 29 .

ADDITIONAL EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the described embodiments, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the embodiments to adapt it to is various usagesand conditions. Thus, other embodiments are also within the claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made to the disclosed embodiments. It is intendedthat the specification and examples be considered as exemplary only,with a true scope of the disclosure being indicated by the followingclaims and their equivalents.

1. A compound of formula (I):

or a salt thereof; wherein

A, a hydrophilic head, is an amino moiety formed from B is C₁-C₂₄ alkyl,C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₄ cycloalkyl, C₁-C₂₄ heteroalkyl,C₁-C₂₄ heterocycloalkyl, aryl, or heteroaryl, or

each occurrence of R₁ and R₂ is independently a C₁-C₂₀ bivalentaliphatic radical; each occurrence of R₃ and R₄, independently, is H orC₁-C₁₀ alkyl, or R₃ and R₄, together with the atom to which they areattached, form C₃-C₁₀ cycloalkyl; each occurrence of R₅ is independentlyC₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₄ cycloalkyl, Cl-C₂₄heteroalkyl, Cl-C₂₄ heterocycloalkyl, aryl, or heteroaryl; eachoccurrence of W is independently O, S, or Se; each occurrence of V isindependently a bond, O, S, or Se; each occurrence of X, a linker, isindependently

in which each occurrence of L₁, L2, L₃, and L₄, independently, is abond, O, S, or NR_(c); each occurrence of G is independently O, S, orNR_(d); each occurrence of Q is independently OR_(f), SR_(g), orNR_(h)R₁; and each occurrence of r and t, independently, is 1-6, eachoccurrence of R_(c), R_(d), R_(f), R_(g), R_(h), and R_(i),independently, is H, C₁-C₁₀ alkyl, C₁-C₁₀ heteroalkyl, aryl, orheteroaryl; and m is independently at each occurrence 0 or 1, providedthat m is 1 when V is S.
 2. (canceled)
 3. The compound of claim 1,wherein B is


4. The compound of claim 1, wherein each X is

and each of R_(c) and R_(d), independently, is H or C₁-C₁₀ alkyl.
 5. Thecompound of claim 1, wherein each of R₁ and R₂ is a C₁-C₄ bivalentaliphatic radical; each of R₃ and R₄, independently, is H or C₁-C₄alkyl; and R₅ is C₁-C₂₀ alkyl.
 6. The compound of claim 1, wherein W isO, S, or Se; and V is a bond.
 7. The compound of claim 1, wherein eachof W and V, independently, is O or Se, and m is
 0. 8. The compound ofclaim 1, wherein each of W and V is O or S; and m is
 1. 9.-16.(canceled)
 17. A compound of formula (I):

or a salt thereof; wherein A, a hydrophilic head, is an amino moietyformed from

B is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₄ cycloalkyl,C₁-C₂₄ heteroalkyl, C₁-C₂₄ heterocycloalkyl, aryl, or heteroaryl, or

each occurrence of R₁ is independently a C₁-C₂₀ bivalent aliphaticradical; each occurrence of R₂ is independently a bond or C₁-C₂₀bivalent aliphatic radical; each occurrence of R₃ and R₄, independently,is H or C₁-C₁₀ alkyl, or R₃ and R₄, together with the atom to which theyare attached, form C₃-C₁₀ cycloalkyl; each occurrence of R₅ isindependently

in which each occurrence of R₆ is independently a bond or C₁-C₂₀bivalent aliphatic radical; each occurrence of R_(b) and R_(b)′ isindependently F or, R_(b) and R_(b)′, together with the atom to whichthey are attached, form C═O; each occurrence of R₇ is F or an aliphaticlipid moiety; each occurrence of L₁ and L₂, independently, is a bond, O,S, or NR_(c), each occurrence of R_(c) is independently H, C₁-C₁₀ alkyl,C₁-C₁₀ heteroalkyl, aryl, or heteroaryl; and n is independently at eachoccurrence an integer from 1 to 20; each occurrence of W and V,independently, is a bond, O, S, or Se; each occurrence of X, a linker,is independently

in which each occurrence of L₃, L₄, L₅, and L₆, independently, is abond, O, S, or NR_(c); each occurrence of G is independently O, S, orNR_(d); each occurrence of Q is independently OR_(f), SR_(g), orNR_(h)R₁; and each occurrence of r and t, independently, is 1-6, eachoccurrence of R_(c), R_(d), R_(e), R_(f), R_(g), R_(h), and R_(i),independently, is H, C₁-C₁₀ alkyl, C₁-C₁₀ heteroalkyl, aryl, orheteroaryl; and m is independently at each occurrence 0 or
 1. 18. Thecompound of claim 17, wherein B is


19. The compound of claim 17, wherein each of R₁ and R₂ is a C₁-C₄bivalent aliphatic radical; and each of R₃ and R₄, independently, is Hor C₁-C₄ alkyl.
 20. The compound of claim 17, wherein each of L₁ and L₂is a bond, and each of R_(b), R_(b)′, and R₇ is F.
 21. The compound ofclaim 17, wherein each of R₂, W, and V is a bond, and m is
 0. 22. Thecompound of claim 19, wherein B is


23. The compound of claim 22, wherein R₁ is a C₁-C₄ bivalent aliphaticradical; and X is

each occurrence of R_(c) and R_(d), independently, is H or C₁-C₁₀ alkyl.24-25. (canceled)
 26. The compound of claim 17, wherein R₆ is C₁-C₄bivalent aliphatic radical; each occurrence of L₁ and L₂, independently,is O or NR_(c), each occurrence of R_(c) is independently H or C₁-C₁₀alkyl; R_(b) and R_(b)′, together with the atom to which they areattached, form C═O; n is independently at each occurrence 1 or 2; and R₇is an aliphatic lipid moiety.
 27. The compound of claim 26, wherein R₇is an aliphatic lipid moiety formed from cholesterol.
 28. The compoundof claim 26, wherein each of R₁ and R₂ is a C₁-C₄ bivalent aliphaticradical; X is

each of R_(c) and R_(d), independently, are H or C₁-C₁₀ alkyl; each of Wand V, independently, is 0, S, or Se; and m is
 0. 29-30. (canceled) 31.A pharmaceutical composition comprising a nanocomplex, wherein thenanocomplex is formed of a compound of claim 1, and a protein or anucleic acid; wherein the nanocomplex has a particle size of 50 nm to1000 nm, and the compound binds to the protein or nucleic acid via anon-covalent interaction, a covalent bond, or both.
 32. Thepharmaceutical composition of claim 31, wherein the protein is GFP-Creor CRISPR/Cas9.
 33. A pharmaceutical composition comprising ananocomplex, wherein the nanocomplex is formed of a compound of claim17, and a protein or a nucleic acid; wherein the nanocomplex has aparticle size of 50 nm to 1000 nm, and the compound binds to the proteinor nucleic acid via a non-covalent interaction, a covalent bond, orboth.
 34. The pharmaceutical composition of claim 33, wherein theprotein is GFP-Cre or CRISPR/Cas9.
 35. A method of treating a medicalcondition, comprising administering to a subject in need thereof aneffective amount of a pharmaceutical composition of claim
 31. 36. Amethod of treating a medical condition, comprising administering to asubject in need thereof an effective amount of a pharmaceuticalcomposition of claim
 33. 37. A pharmaceutical composition comprising ananocomplex, wherein the nanocomplex is formed of a compound of claim 1,and a small molecule; wherein the nanocomplex has a particle size of 50nm to 1000 nm, and the compound binds to the small molecules via anon-covalent interaction, a covalent bond, or both.
 38. Thepharmaceutical composition of claim 37, wherein the small molecule is anantifungal agent or a chemotherapeutic agent.
 39. The pharmaceuticalcomposition of claim 37, wherein the small molecule is selected from thegroup consisting of Bortezomib, Imatinib, Gefitinib, Erlotinib,Afatinib, Osimertinib, Dacomitinib, Daunorubicin hydrochloride,cytarabine, Fluorouracil, Irinotecan Hydrochloride, Vincristine Sulfate,Methotrexate, Paclitaxel, Vincristine Sulfate, epirubicin, docetaxel,Cyclophosphamide, Carboplatin, Lenalidomide, Ibrutinib, Abirateroneacetate, Enzalutamide, Pemetrexed, Palbociclib, Nilotinib, Everolimus,Ruxolitinib, epirubicin, pirirubicin, idarubicin, valrubicin, amrubicin,Bleomycin, phleomycin, dactinomycin, Mithramycin, streptozotecin,pentostatin, Mitosanes mitomycin C, Enediynes calicheamycin, Glycosidesrebeccamycin, Macrolide lactones epotihilones, ixabepilone, pentostatin,Salinosporamide A, Vinblastine, Vincristine, Etoposide, Teniposide,Vinorelbine, Docetaxel, Camptothecin, Hycamtin, Pederin, Theopederins,Annamides, Trabectedin, Aplidine, and Ecteinascidin 743 (ET743).
 40. Thepharmaceutical composition of claim 37, wherein the small molecule isAmphotericin B or Doxorubicin.
 41. A pharmaceutical compositioncomprising a nanocomplex, wherein the nanocomplex is formed of acompound of claim 17 , and a small molecule; wherein the nanocomplex hasa particle size of 50 nm to 1000 nm, and the compound binds to the smallmolecule via a non-covalent interaction, a covalent bond, or both. 42.The pharmaceutical composition of claim 41, wherein the small moleculeis an antifungal agent or a chemotherapeutic agent.
 43. Thepharmaceutical composition of claim 41, wherein the small molecule isselected from the group consisting of Bortezomib, Imatinib, Gefitinib,Erlotinib, Afatinib, Osimertinib, Dacomitinib, Daunorubicinhydrochloride, cytarabine, Fluorouracil, Irinotecan Hydrochloride,Vincristine Sulfate, Methotrexate, Paclitaxel, Vincristine Sulfate,epirubicin, docetaxel, Cyclophosphamide, Carboplatin, Lenalidomide,Ibrutinib, Abiraterone acetate, Enzalutamide, Pemetrexed, Palbociclib,Nilotinib, Everolimus, Ruxolitinib, epirubicin, pirirubicin, idarubicin,valrubicin, amrubicin, Bleomycin, phleomycin, dactinomycin, Mithramycin,streptozotecin, pentostatin, Mitosanes mitomycin C, Enediynescalicheamycin, Glycosides rebeccamycin, Macrolide lactones epotihilones,ixabepilone, pentostatin, Salinosporamide A, Vinblastine, Vincristine,Etoposide, Teniposide, Vinorelbine, Docetaxel, Camptothecin, Hycamtin,Pederin, Theopederins, Annamides, Trabectedin, Aplidine, andEcteinascidin 743 (ET743).
 44. The pharmaceutical composition of claim41, wherein the small molecule is Amphotericin B or Doxorubicin.
 45. Amethod of treating a medical condition, comprising administering to asubject in need thereof an effective amount of a pharmaceuticalcomposition of claim
 37. 46. A method of treating a medical condition,comprising administering to a subject in need thereof an effectiveamount of a pharmaceutical composition of claim 41.